Mzb1, a novel b cell factor, and uses thereof

ABSTRACT

The marginal zone (MZ) and B1 subsets of B cells, which differ from conventional follicular (FO) B cells both developmentally and functionally, are involved in early responses to infectious pathogens and the production of self-reactive antibodies. A novel gene, mzb1, is expressed at high levels in MZ and B1 B cells but at low level, if at all, in FO B cells. MZB1 is involved in the regulation of proliferation, BCR-mediated signal transduction, and antibody production in B cells. Inhibitors, activators and enhancers of MZB1 expression or activity can be used as immune modulators for research and therapeutic purposes.

FIELD OF THE INVENTION

The present invention relates to the field of immunology, in particular,the field of B cell biology. The present invention provides a novel Bcell factor, MZB1, which is involved in the regulation of B cellreceptor (BCR)-mediated signaling and antibody production. The presentinvention further provides agents which modulate the expression and/orthe activity of MZB1 and the use of these agents for research andtherapeutic purposes.

BACKGROUND OF THE INVENTION

The vertebrate immune system protects the host from foreign pathogenssuch as virus, bacteria, parasite and fungus as well as senesced,damaged or diseased host cells. The host defense against foreignpathogens can be divided into three phases: innate immunity, “naturalmemory” and adaptive immunity.

Innate immunity is the first line of defense against foreign pathogens,which is active within minutes or hours after exposure to pathogen. Theinnate immunity is not antigen-specific; it is carried out by immunecells such as macrophages, mast cells, granulocytes (basophils,eosinophils, neutrophils), natural killer cells, and γδT cells, whichrecognize features that are common to many pathogens rather thanspecific antigens.

In contrast, the adaptive immunity is antigen-specific; it is carriedout by antigen-specific T cells and B cells, in particular, follicular(FO) B cells, and takes days to two weeks to develop. T cells and FO Bcells express antigen-specific receptors, T cell receptor (TCR) and Bcell receptor (BCR), respectively, which are encoded by genes rearrangedfrom the germline conformation.

In addition to being antigen-specific, the adaptive immunity differsfrom the innate immunity in its capability of generating immunologicalmemory. The memory B and T cells are capable of launching a more rapidand more robust response against foreign antigens upon re-exposure tothe same antigens.

The natural memory bridges the temporal gap between the innate immuneresponse and the adaptive immune response; it is carried out by severalcomponents of the B and T cell lineages but does not generate lastingprotective immunity for longer than a few days.

An important part of the natural memory immune response is theproduction of natural antibodies which have high cross-reactivity butlow binding affinity against both microbial agents and some selfantigens (Baumgarth et al., 1999; Baumgarth et al., 2000; Boes et al.,1998; Ochsenbein et al., 1999). The natural antibodies are encoded byrearranged antibody genes that have not undergone somatic mutation andare produced by marginal zone (MZ) B cells and peritoneal B1 cells. MZ Bcells play a key role in the early response to pathogens in thebloodstream, whereas B1 cells in the pleural and peritoneal cavity playa key role in the response to pathogens introduced in the mucosalsurfaces (see review by Lopes-Carvalho T and Kearney J F, 2004). Recentstudies have suggested that B1 B cells and MZ B cells are also a sourceof auto-antibodies.

Even though B1 B cells and MZ B cells play a key role in natural memoryimmune response against foreign pathogens and are likely involved inautoimmune diseases involving auto-antibodies, the development,homeostasis and activation of these B cell subsets are only beginning tobe elucidated (see reviews by Martin F and Kearney J F, 2000 andSrivastava B et al., 2005). Therefore, there remains a need in the artto better understand the biology of MZ and B1 B cells, in particular,their activation and the regulation of natural antibody production.Furthermore, there remains a need in the art to develop tools whichallow for the modulation of the activities of MZ and B1 B cells, inparticular, the production of natural antibodies, more particularly, theproduction of auto-reactive antibodies.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

-   (a) a nucleotide sequence as set forth in SEQ ID NO:1, 3, 11, 12 or    13,-   (b) a nucleotide sequence encoding a polypeptide having an amino    acid sequence as set forth in SEQ ID NO:2 or 4,-   (c) a nucleotide sequence encoding a polypeptide having an amino    acid sequence as set forth in SEQ ID NO:2 or 4 further comprising an    amino-terminal methionine,-   (d) a nucleotide sequence that is an ortholog of any of (a)-(c),-   (e) a nucleotide sequence that is an allelic variant or a splice    variant of any of (a)-(d),-   (f) a nucleotide sequence that is at least 50% identical to any of    (a)-(e) over its entire length,-   (g) a nucleotide sequence that hybridizes under highly stringent or    moderately stringent conditions to any of (a)-(e),-   (h) a nucleotide sequence encoding a polypeptide having an amino    acid sequence as set forth in SEQ ID NO:2 or 4 with at least one    amino acid modification selected from substitution, insertion,    deletion, amino terminal turnction, carboxyl terminal truncation, or    any combination thereof,-   (i) a nucleotide sequence comprising at least 12 consecutive    nucleotides of any of (a)-(h), and-   (j) a nucleotide sequence that is complementary to any of (a)-(i),    wherein (d)-(g) encodes a polypeptide having at least one of the    biological activities of the polypeptide encoded by any of (a)-(c).

The present invention also provide n isolated polypeptide comprising anamino acid sequence selected from the group consisting of:

-   (a) the amino acid sequence as set forth in SEQ ID NO:2 or 4,-   (b) an amino acid sequence encoded by the nucleotide sequence set    forth in SEQ ID NO:1 or 3,-   (c) an amino acid sequence of (a) or (b) further comprising an    amino-terminal methionine,-   (d) an amino acid sequence which is an ortholog any of (a)-(c),    optionally further comprising an amino-terminal methionine,-   (e) an amino acid sequence which is an allelic variant or a splice    variant of any of (a)-(d), optionally further comprising an    amino-terminal methionine,-   (f) an amino acid sequece which is at least 72% identical to any of    (a)-(e) over its entire length, optionally further comprising an    amino-terminal methionine,-   (g) an amino acid sequence as set forth in SEQ ID NO:2 or 4 with at    least one amino acid modification selected from substitution,    insertion, deletion, amino terminal turnction, carboxyl terminal    truncation, or any combination thereof, optionally further    comprising an amino-terminal methionine,-   (h) an amino acid sequence comprising at least 10 consecutive amino    acids of any of (a)-(g), optionally further comprising an    amino-terminal methionine,-   (j) an amino acid sequence encoded by the isolated nucleic acid of    any of claims 1(a)-(i), optionally further comprising an    amino-terminal methionine, and-   (k) an amino acid sequence comprising any of (a)-(j) fused to a    heterologous sequence,    wherein (d)-(f) has at least one of the biological activities of the    polypeptide having the amino acid sequence of any of (a)-(c).

The present invention further provides a vector comprising an isolatednucleic acid of the present invention operably linked to atranscriptional regulatory sequence.

The present invention additionally provides a host cell or a hostorganism comprising an isolated nucleic acid or a vector of the presentinvention.

The present invention provides an antibody or an antigen-bindingfragment thereof which specifically binds a polypeptide of the presentinvention and a hybridoma or a host cell producing said antibody.

The present invention also provides a process for producing apolypeptide of the present invention, comprising culturing the host cellof the present invention under suitable conditions to express thepolypeptide, and optionally isolating the polypeptide from the culture,wherein the expression vector contained in the host cell comprises anucleic acid of any of (a)-(i) described above.

The present invention provides an inhibitor of the expression or atleast one of the biological activities of a polypeptide of the presentinvention which is biologically active.

The present invention also provides an activator or enhancer of theexpression or at least one of the biological activities of thepolypeptide of the present invention which is biologically active.

The present invention further provides a pharmaceutical compositioncomprising an inhibitor or an activator or enhancer of the presentinvention.

The present invention provides the use of an inhibitor of the presentinvention for treating an autoimmune disease. Thus, the presentinvention provides remedies for autoimmune diseases, in particular,means, methods and uses for preventing and/or treating autoimmunediseases.

The present invention also provides the use of an activator or enhancerof the present invention for treating an immunodeficiency. Thus, thepresent invention provides remedies for immunodeficiency, in particular,means, methods and uses for preventing and/or treating immunodeficiency.

The present invention additionaly provides an in vitro method forenhancing the antibody production in a cell, comprising the step ofcontacting the cell with an activator or enhancer of the presentinvention, wherein the cell is capable of producing an antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Expression pattern of MZB1.

(A) MZB1 expression is restricted to B cell tissues. Poly-A⁺ RNA (2 μg)from adult mice and 5 μg of cytoplasmic RNA from the pre-B cell linePD36 were utilized for an RNA analysis to detect MZB1 specifictranscripts. As loading control and control for B cell contamination,probes hybridizing the constant region of Igκ and GADPH were utilized.

(B) MZB1 expression in different B cell populations. Western blot wasperformed on 30 μg of total protein extract from different FACS-sorted Bcell populations.

FIG. 2: Indirect immunofluorescence (IF) on FACS-sorted marginal zone(MZ) B cells. The isolated cells were fixed with 4% paraformaldehyde andused for IF experiments with rat anti-MZB1 followed by anti-rat Alexa647antibodies.

FIG. 3: Indirect immunofluorescence (IF) on NIH 3T3 cells stablyexpressing FLAG-MZB1. The cells were fixed with 4% paraformaldehyde andused for IF-experiments with rat α-MZB1 followed by α-rat Alexa633 (red)and/or α-BAP31 followed by α-mouse Alexa488 (green) antibodies.

FIG. 4: Indirect immunofluorescence (IF) on NIH3T3 cells stablytransfected with either MZB1-GFP or ΔN-MZB1-GFP. NIH3T3 fibroblasticcells were stably transfected with either MZB1-GFP (MZB1-GFP fusionprotein) or ΔN-MZB1-GFP (MZB1-GFP fusion protein lacking the N-terminalsignal peptide of MZB1). Cells were treated for 6 h with DMSO (control)or 1 μg/ml nocodazole. The treated cells were fixed with 4%paraformaldehyde and used for IF-experiments with rat αMZB1 followed byαrat Alexa647 antibodies (green) and rabbit αBAP31 followed by αrabbitAlexa568 antibodies (red). BAP31 was used as a marker for ER membranes.

FIG. 5: Preparation of endoplasmatic reticulum (ER) vesicles andproteinase K digestion. K46 cells were resuspended in a hypertonicbuffer containing 250 mM sucrose, and homogenized by passing through a22 gauge needle 15 times and centrifuged at 3000 g for 10 min. Thesupernatant was centrifuged at 20,000 g for 15 min. and the resultingpellet, containing mitochondrial membranes, was discarded. The resultingsupernatant (S-20) was centrifuged at 100,000 g for 1 h. The pelletcontained sealed cytosolic-side-out ER vesicles (microsomes), which wereresuspended in the hypertonic lysis buffer plus 100 mM NaCl. Aliquots ofthe preparation (−10 μg of protein), S-100 and microsomal fraction, weretreated with 2 μg/μl proteinase K in the presence or absence of 1%Triton X-100 in a total volume of 12 μl for 30 min. on ice. The reactionwas stopped by adding PMSF at a final concentration of 30 mM and sampleswere subjected to TCA-precipitation followed by SDS-PAGE and immunoblotanalysis using antibodies specific for MZB1. As a control for the ERvesicle preparation, additional immunoblot analysis using antibodiesspecific for the mitochondrial transmembrane protein BAP32, the ERtransmembrane proteins BAP31 and Calnexin, the luminal ER protein BiP,the ER membrane-associated, cytosolic protein RACK1 as well as thecytosolic protein GAPDH were performed.

FIG. 6: Immunoblot analysis with antibodies specific for MZB1, BAP32 andGAPDH was performed on 10 μg of cytosolic and membrane fractionsextracted from K46 mature B cells.

FIG. 7: Immunoblot analysis with antibodies specific for MZB1, BAP32 andGAPDH was performed on 10 μg of cytosolic and membrane fractionsextracted from non-stimulated and stimulated K46 mature B cells. Cellswere BCR stimulated with 5 μg/ml α-kappa antibody for the indicated timespans.

FIG. 8: Recruitment of MZB1 to the cell membrane following BCRstimulation of K46 B cells. Densitometric analysis of MZB1-specificimmunoblot signals depicted in FIG. 7.

FIG. 9: Identification of the receptor for activated C kinase 1 (RACK1)as an MZB1 interaction partner in the membrane fraction of K46 B cells.

(A) Anti-RACK1 Immunoprecipitation (IP). Membrane and cytosol fractionsof K46 B cells were prepared. Immunoprecipitation (IP) was performedusing anti-RACK1 and/or Protein L beads (ctrl.) utilizing the cytosolicor membrane fractions. The presence of MZB1 and RACK1 was monitored byimmunoblotting.

(B) Anti-MZB1 Immunoprecipitation (IP). Membrane and cytosol fractionsof K46 B cells were prepared. Immunoprecipitation (IP) was performedusing anti-MZB1 sepharose beads and the cytosolic or membrane fractions.The presence of MZB1 and RACK1 was monitored by immunoblotting.

FIG. 10: Interaction of RACK1, PKCβ and MZB1 in the membrane fraction ofK46 B cells. Anti-PKCβ Immunoprecipitation (IP). Membrane and cytosolfractions of K46 B cells were prepared. Immunoprecipitation (IP) wasperformed using anti-PKCβ and Protein G beads utilizing the cytosolic ormembrane fractions. Protein G beads alone served as the control. Thepresence of MZB1, PKCβ and RACK1 was monitored by immunoblotting.

FIG. 11: MZB1 complex purification. Approximately 1.0 I of K46 mature Bcells were cultured until reaching the exponential growth phase,harvested and in vivo formaldehyde crosslinking was performed by adding1% formaldehyde (final concentration) directly to the K46 cell culture.Cells were incubated for 10 min at 37° C. and 5% CO₂ and thecrosslinking reaction was quenched by addition of 2.5M glycine with afinal concentration of 125 mM. After crosslinking, cells were collected,washed with ice-cold PBS and frozen at −80° C. prior to lysis. Aftercell extract preparation, using an SDS containing hypotonic lysis bufferand extensive sonication, MZB1 was immunoprecipitated using anMZB1-specific antibody, directly coupled to 4B-sepharose beads. Ascontrols, MZB1 was immunoprecipitated from un-crosslinked K46 cellextract, and, in addition, 4B beads were incubated with crosslinked K46cell extract. The immunoprecipitated proteins were eluted with samplebuffer and separated on a 4%-12% gradient SDS-PAGE. The gel was silverstained. The marked bands (arrow) were cut out and prepared for massspectrometry analysis.

FIG. 12: Verification of ERp57, GRP94 (Endoplasmin), ERp44 and BiP aspotential MZB1 interaction partners. Anti-MZB1 Immunoprecipitation (IP).Cellular extracts of K46 B cells were prepared. Immunoprecipitation (IP)was performed using anti-MZB1 sepharose beads and anti-EBNA sepharose ascontrol. The presence of MZB1, ERp57, GRP94, ERp44 and BiP was monitoredby immunoblotting.

FIG. 13: Verification of ERp57 and GRP94 (Endoplasmin) as potential MZB1interaction partners.

(A) Anti-ERp57 Immunoprecipitation (IP). Cellular extracts of K46 Bcells were prepared. Immunoprecipitation (IP) was performed usinganti-ERp57 antibodies in combination with Protein G beads or Protein Gbeads alone (control). The presence of MZB1 and ERp57 was monitored byimmunoblotting.

(B) Anti-GRP94 Immunoprecipitation (IP). Cellular extracts of K46 Bcells were prepared. Immunoprecipitation (IP) was performed usinganti-GRP94 antibodies in combination with Protein G beads or Protein Gbeads alone (control). The presence of MZB1 and GRP94 was monitored byimmunoblotting.

FIG. 14: MZB1 interacts with ERp57, but not GRP94 (Endoplasmin), in aCa²⁺-dependent fashion. Anti-MZB1 Immunoprecipitation (IP). Cellularextracts of K46 B cells were prepared in the presence or the absence of2.5 mM Ca²⁺. Immunoprecipitation (IP) was performed using anti-MZB1sepharose beads and anti-EBNA sepharose as control. The presence ofMZB1, ERp57, GRP94, ERp44 and BiP was monitored by immunoblotting.

FIG. 15: MZB1 interacts with ERp57, but not GRP94 (Endoplasmin), in aCa²⁺-dependent fashion.

(A) Anti-ERp57 Immunoprecipitation (IP). Cellular extracts of K46 Bcells were prepared in the presence or the absence of 2.5 mM Ca²⁺.Immunoprecipitation (IP) was performed using anti-ERp57 antibodies incombination with Protein G beads or Protein G beads alone (control). Thepresence of MZB1 and ERp57 was monitored by immunoblotting.

(B) Anti-GRP94 Immunoprecipitation (IP). Cellular extracts of K46 Bcells were prepared in the presence or the absence of 2.5 mM Ca²⁺.Immunoprecipitation (IP) was performed using anti-GRP94 antibodies incombination with Protein G beads or Protein G beads alone (control). Thepresence of MZB1 and GRP94 was monitored by immunoblotting.

FIG. 16: Size exclusion chromatography of the endogenous MZB1, ERp57,GRP94 (Endoplasmin), ERp44 and Bip proteins in the membrane fraction ofK46 cells. 500 μg membrane protein extract of K46 cells was preparedeither in the presence of Ca²⁺ (2.5 mM CaCl₂) or in its absence (10 mMEDTA), and the extracts were separated on an analytical HR10/30 Superdex200 (Amersham/GE Healthcare) column. The immunoblot (IB) analysis wasperformed according to standard procedures.

FIG. 17: FACS analysis of integrin and TLR4 surface expression onMZB1-transduced FO B cells. FACS-sorted FO B cells(B220⁺CD21^(int)CD23^(hi)) were either retrovirally transduced with GFPalone (MOCK) or with MZB1 and GFP (+MZB1). The transduced FO B cellswere gated on their GFP⁺ populations (data not shown), which werefurther analyzed for their integrin surface expression (A) (β1, β2 andβ7 integrins) (A) or their TLR4 surface expression (B), respectively.For the analysis of TLR4 expression, cells were either kept unstimulatedor stimulated with 10 μg/ml LPS for 12 hours. Numbers indicatepercentages of surface-integrin (A) or surface TLR4 (B) positive cells.

FIG. 18: FACS analysis of integrin surface expression on K46 MZB1siRNAcells. K46 wt, K46 siRNA and K46 siRNA::MZB1 cells were gated on theirGFP⁺ populations (data not shown), which were further analyzed for theirintegrin surface expression (α4, β1 and β2 integrins).

FIG. 19: Size exclusion chromatography of endogenous MZB1 protein in thecytosolic fraction of K46 cells. 500 μg cytosolic protein extract of K46cells was prepared either in the presence of Ca²⁺ (2.5 mM CaCl₂ or inits absence (10 mM EDTA), and the extracts were separated on ananalytical HR10/30 Superdex 200 (Amersham/GE Healthcare) column. Theimmunoblot (IB) analysis was performed according to standard procedures.

FIG. 20: Size exclusion chromatography of endogenous MZB1 protein in themembrane fraction of K46 cells. 500 μg membrane protein extract of K46cells was prepared either in the presence of Ca²⁺ (2.5 mM CaCl₂ or inits absence (10 mM EDTA), and the extracts were separated on ananalytical HR10/30 Superdex 200 (Amersham/GE Healthcare) column. Theimmunoblot (IB) analysis was performed according to standard procedures.

FIG. 21: Downregulation of MZB1 expression using shRNA. Immunoblotananlysis with MZB1- and GAPDH-specific antibodies was performed on 20μg of total protein extract from wt, shRNA and shRNA+MZB1 K46 cells.

FIG. 22: Restoration of MZB1 expression in shRNA K46 cells: generationof shRNA+MZB1 K46 cells.

FIG. 23: Proliferation of wt, shRNA and shRNA+MZB1 K46 B cells. Cellswere left untreated and cell numbers were determined using a CASY® cellcounter (CASY®-technology) after 0 hours, 26 hours and 72 hours afterplating. Data are expressed as the mean cell numbers of triplicatecultures.

FIG. 24: Proliferation of wt, shRNA and shRNA+MZB1 K46 B cells. Cellswere left untreated or treated with anti-κ (5 μg/ml) and pulsed with 1.5μCi/well [³H] thymidine for 16 h before being collected. Data areexpressed as the mean [³H] thymidine incorporation of triplicatecultures.

FIG. 25: GFP expression in wt and shRNA K46 B cells.

FIG. 26: Immunoblot ananlysis with MZB1- and GAPDH-specific antibodieswas performed on 20 μg of total protein extract from wt, shRNA-GFP-intand shRNA-GFP-high K46 cells.

FIG. 27: Proliferation of wt, shRNA-GFP-int and shRNA-GFP-high K46 Bcells. Cells were left untreated or treated with anti-κ (5 μg/ml) andpulsed with 1.5 μCi [³H] thymidine for 16 h before being collected. Dataare expressed as the mean [³H] thymidine incorporation of triplicatecultures.

FIG. 28: Annexin V-PE Staining. K46-wt or K46-shRNA cells were washedwith cold PBS and incubated with Annexin V-PE in a buffer containing7-amino-actinomycin D (7AAD). After incubating the cells at RT in thedark, the samples were analyzed by flow cytometry on a FACSCalibur flowcytometer.

FIG. 29: Downregulation of MZB1 in K46 mature B cells changes BCRsignaling capacity. Ca²⁺ mobilization upon BCR engagement in wt, shRNAand shRNA+MZB1 K46 cells. Cells were stimulated with anti-κ (5 μg/ml) in0.5 mM Ca²⁺ containing media and increases in free intracellular Ca²⁺were measured in real time using a FACSAria Flow-Cytometer.

FIG. 30: Downregulation of MZB1 in K46 mature B cells changes BCRsignaling capacity. Ca²⁺ mobilization upon BCR engagement in shRNA andshRNA+MZB1 K46 cells. Cells were stimulated with anti-κ (5 μg/ml) in 2.0mM Ca²⁺containing media and increases in free intracellular Ca²⁺ weremeasured in real time using a FACSAria Flow-Cytometer.

FIG. 31: Surface BCR expression in wt and shRNA K46 B cells. Cells werestained with kappa light chain-specific antibodies and subjected to FACSanalysis. Cells were gated for GFP⁺.

FIG. 32: MZB1 regulates Ca²⁺ flux in K46 mature B cells. Measurement ofCa²⁺ flux of Indo-1 AM loaded K46 B cells (wt), K46-MZB1-siRNA cells andK46-MZB1-siRNA cells with restored MZB1-expression after treatment with1 μM thapsigargin. Cells were placed in medium where extracellular Ca²⁺was chelated by EGTA, and after addition of 1 μM thapsigargin, 5 mM ofCa²⁺ was added at the indicated time point. Increases in freeintracellular Ca²⁺ were measured in real time using a FACSAriaFlow-Cytometer.

FIG. 33: MZB1 redistributes into punctuate structures after ER Ca²⁺store depletion. NIH 3T3 fibroblastic cells were stably transfected withMZB1-GFP and plated out in phenolred-free Dulbecco's Modified EagleMedium low glucose supplemented with 10% FCS, 2 mM L-Glutamine as wellas 0.1 mg/ml Streptomycin and 10 U/ml Penicillin in ibidi 35 mm highp-dishes at a density of 2.5×10″ cells per ml. The next day, dishes weresubjected to confocal imaging analysis (Leica Confocal) before (0 min)and after (30 min) addition of 1 μM thapsigargin and 3 mM EGTA.

FIG. 34: MZB1 redistributes into punctuate structures after ER Ca²⁺store depletion. NIH 3T3 fibroblastic cells were stably transfected withMZB1-GFP and plated out in phenolred-free Dulbecco's Modified EagleMedium low glucose supplemented with 10% FCS, 2 mM L-Glutamine as wellas 0.1 mg/ml Streptomycin and 10 U/ml Penicillin in ibidi 35 mm highμ-dishes at a density of 2.5×10⁴ cells per ml. The next day, dishes weresubjected to confocal imaging analysis (Leica Confocal) before (0 min)and after (30 min) addition of DMSO.

FIG. 35: MZB1 regulates ER-Ca²⁺ stores in K46 mature B cells.Measurement of Ca²⁺ flux of Indo-1 AM loaded K46 B cells (wt),K46-MZB1-siRNA cells and K46-MZB1-siRNA cells with restoredMZB1-expression after treatment with 1.4 μM ionomycin. Cells were placedin medium containing 2.5 mM Ca²⁺ followed by the simultaneous additionof 1 μM thapsigargin and 3 mM EGTA at the indicated time point.Increases in free intracellular Ca²⁺ were measured in real time using aFACSAria Flow-Cytometer.

FIG. 36: MZB1 increases ER-Ca²⁺ stores in MZB1-transduced FO B cells.

(A) FACS-sorted FO B cells (B220⁺CD21^(int)CD23^(hi)) were eitherretrovirally transduced with GFP alone (MOCK) or with MZB1 and GFP(+MZB1). Transduced, GFP⁺ FO B cells were placed in medium containing 2mM Ca²⁺ followed by the simultaneous addition of 1.4 μM thapsigargin and3 mM EGTA at the indicated time point. Increases in free intracellularCa²⁺ were measured in real time using a FACSAria Flow-Cytometer. Thefigure displays the gate for transduced, GFP⁺ cells.

(B) FACS-sorted FO B cells (B220⁺CD21^(int)CD23^(hi)) were eitherretrovirally transduced with GFP alone (MOCK) or with AL1 and GFP(+AL1). Transduced, GFP⁺ FO B cells were enriched by FACS-sorting. Animmunoblot analysis with AL1-specific antibodies (clone 2F9) wasperformed on 20 mg total protein extracts from splenic FO B cells,infected with the AL1- and GFP-expressing (+AL1) or the solelyGFP-expressing retrovirus (MOCK). As a control for loading and transfer,an additional immunoblot analysis using a GAPDH-specific antibody wasperformed.

FIG. 37: Genetic inactivation of the mzb1 locus via homologousrecombination. Neo: neomycin phosphotransferase gene.

FIG. 38: Secretion of IgM antibodies in follicular B cellsoverexpressing MZB1 and stimulated with LPS. Primary follicular B cellswere infected with an MZB1-expressing retrovirus pEG2-MZB1 and an emptyvector control retrovirus pEG2-MCS. Infected cells were stimulated with1.0 μg/ml LPS. IgM concentration in the cell culture supernatant wasdetermined by ELISA 6 hours, 1 day, 2 days, and 3 three dayspost-stimulation.

FIG. 39: MZB1 expression in different T cell populations derived fromtransgenic mice. An immunoblot analysis with MZB1-specific antibodies(clone 2F9) was performed on 30 μg total protein extracts from thymusderived double negative (DN; CD4⁻ and CD8⁻) T cell progenitors, thymusderived double positive (DP; CD4⁺ and CD8⁺) T cell progenitors, CD4⁺ andCD8⁺ single positive thymocytes, and in addition, from CD4⁺ as well asCD8⁺ single positive splenic and lymph node derived peripheral T cells.All cells analyzed were isolated from 5-9 weeks old transgenic mice. Asa control for loading and transfer an additional immunoblot analysisusing a GAPDH-specific antibody was performed.

FIG. 40: MZB1 expression in different T cell populations derived fromtransgenic mice and their corresponding wt littermates. An immunoblotanalysis with MZB1-specific antibodies (clone 2F9) was performed on 30μg total protein extracts from thymus-, lymph node-, and spleen-derivedCD4⁺ as well as CD8⁺ single positive T cells (mixture of CD4⁺ and CD8⁺single positive cells). All cells analyzed were isolated from 5-9 weeksold transgenic mice or their corresponding wt littermates. As a controlfor loading and transfer an additional immunoblot analysis using aGAPDH-specific antibody was performed.

FIG. 41: Measurement of endogenous ceramide levels in CD4⁺ splenic Tcells derived from transgenic mice and their corresponding wtlittermates. FACS-sorted CD4⁺ splenocytes, either isolated from 5-9weeks old transgenic mice or their corresponding wt littermates wereleft untreated or induced with α-CD28 (0.1 μg/ml, 0.3 μg/ml; or 1.0μg/ml), α-CD3ε (0.3 μg/ml) or both for 36 hours. Cells were harvestedand endogenous ceramide was extracted. The isolatedsphingomyelin-derived ceramide was radioactively labeled by performing aDAG kinase assay using [γ-³²P]ATP. The resulting [³²P]ceramide wasquantified using a phosphor-imager system (Fuji) and theAlphaImager™-quantification software (Alpha Innotech) followingthin-layer chromatography (TLC). Data are expressed as the meanintensity of [³²P]ceramide-spots on the TLC plates of duplicatecultures. The figure is a representative of three independentexperiments.

FIG. 42: Measurement of endogenous ceramide levels in wt fibroblastscompared to fibroblast cells stably expressing MZB1. Non-stimulated (Ø)NIH 3T3 cells (NIH3T3) and NIH 3T3 cells stably transfected withFLAG-MZB1 (NIH3T3::MZB1) were grown to confluency, cells were harvestedand endogenous ceramide was extracted. The isolated ceramide wasradioactively labeled by performing a DAG kinase assay using [γ-³²P]ATP.The resulting [³²P]ceramide was quantified using a phosphor-imagersystem (Fuji) and the AlphaImager™-quantification software (AlphaInnotech) following thin-layer chromatography (TLC). Data are expressedas the mean intensity of [³²P]ceramide-spots on the TLC plates ofduplicate cultures. The figure is a representative of three independentexperiments.

FIG. 43: Measurement of endogenous A-SMase activity in CD4⁺ splenic Tcells derived from transgenic mice and their corresponding wtlittermates. MACS®-purified CD4⁺ splenocytes, either isolated from 5-9weeks old transgenic mice or their corresponding wt littermates wereleft untreated or induced with both α-CD28 (1.0 μg/ml) and α-CD3ε (0.3μg/ml) for 36 hours. Cells were harvested, lysed and the cellularextracts (50 μg total protein) were incubated in the appropriate bufferconditions, being specific for acid-SMases (pH 5.0). Prior toincubation, [N-methyl-¹⁴C]sphingomyelin (Amersham/GE Healthcare) asradioactively labeled substrate was added. The amount of C¹⁴-labeledphosphorylcholine produced by A-SMases from [¹⁴C]sphingomyelin wasmeasured by scintillation counting. Data are expressed as the meanc.p.m. ([¹⁴C]phosphorylcholine) of duplicate cultures. As a control,samples without cellular extracts (w/o enzyme) and samples withoutradioactively labeled substrate (w/o enzyme) were performed. The figureis a representative of three independent experiments.

FIG. 44: Measurement of endogenous N-SMase activity in CD4⁺ splenic Tcells derived from transgenic mice and their corresponding wtlittermates. MACS®-purified CD4⁺ splenocytes, either isolated from 5-9weeks old transgenic mice or their corresponding wt littermates wereleft untreated or induced with both α-CD28 (1.0 μg/ml) and α-CD3ε (0.3μg/ml) for 36 hours. Cells were harvested, lysed and the cellularextracts (50 μg total protein) were incubated in the appropriate bufferconditions, being specific for neutral-SMases (pH 7.4). Prior toincubation, [N-methyl-¹⁴C]sphingomyelin (Amersham/GE Healthcare) asradioactively labeled substrate was added. The amount of C¹⁴-labeledphosphorylcholine produced by N-SMases from [¹⁴C]sphingomyelin wasmeasured by scintillation counting. Data are expressed as the meanc.p.m. ([¹⁴C]phosphorylcholine) of duplicate cultures. As a control,samples without cellular extracts (w/o enzyme) and samples withoutradioactively labeled substrate (w/o enzyme) were performed. The Figureis a representative of three independent experiments.

FIG. 45: Measurement of [³H]thymidine incorporation in CD4⁺ lymphnode-derived T cells isolated from transgenic mice and theircorresponding wt littermates. LN-derived, FACS-sorted CD4⁺ T cells,either isolated from 5-9 weeks old transgenic mice or theircorresponding wt littermates were plated in a 96 well format. Cells wereeither left untreated or induced with α-CD28 (0.1 μg/ml), α-CD3ε (0.3μg/ml) or both for 12 hours. Each well was pulsed with 1.5 μCi[³H]thymidine 12 h-16 h before being collected. Data are expressed asthe mean [³H]thymidine incorporation of triplicate cultures. The figureis a representative of three independent experiments.

FIG. 46: Measurement of [³H]thymidine incorporation in CD4⁺ splenic Tcells isolated from transgenic mice and their corresponding wtlittermates. FACS-sorted splenic CD4⁺ T cells, either isolated from 5-9weeks old transgenic mice or their corresponding wt littermates wereplated in a 96 well format. Cells were either left untreated or inducedwith α-CD28 (0.1 μg/ml), α-CD3ε (0.3 μg/ml) or both for 12 hours. Eachwell was pulsed with 1.5 μCi [³H]thymidine 12 h-16 h before beingcollected. Data are expressed as the mean [³H]thymidine incorporation oftriplicate cultures. The figure is a representative of three independentexperiments.

FIG. 47: Measurement of [³H]thymidine incorporation in CD4⁺ lymphnode-derived and in CD4⁺ splenic T cells isolated from transgenic miceand their corresponding wt littermates. LN-derived (A), and splenic (B)FACS-sorted CD4⁺ T cells, either isolated from 5-9 weeks old transgenicmice or their corresponding wt littermates were plated in a 96 wellformat. Cells were either left untreated or induced with α-CD28 (0.3μg/ml), α-CD3ε (0.3 μg/ml) or both for 12 hours. Each well was pulsedwith 1.5 μCi [³H] thymidine 12 h-16 h before being collected. Data areexpressed as the mean [³H] thymidine incorporation of triplicatecultures. The figure is a representative of three independentexperiments.

FIG. 48: TUNEL assay on CD4⁺ splenic T cells isolated from transgenicmice and their corresponding wt littermates. FACS-sorted CD4⁺splenocytes, either isolated from 5-9 weeks old transgenic mice or theircorresponding wt littermates were TCR-stimulated with α-CD3ε (0.3 μg/ml)alone, or with both α-CD28 (0.1 μg/ml) and α-CD3ε (0.3 μg/ml) for 24hours at 37° C. in a 5% CO₂-humidified incubator. For determining theapoptotic status, the cells were stained with the “In Situ Cell DeathDetection Kit” (Roche) according to the manufacturers instructions andthe levels of APC⁺ apoptotic cells were quantified by flow cytometry.Percentages of apoptotic cells staining positive for APC-dUTP are shown.Negative controls are shown in red. The figure is a representative ofthree independent experiments.

FIG. 49: Calcium responses in CD4⁺ splenic T cells isolated fromtransgenic mice and their corresponding wt littermates. MACS®-purifiedCD4⁺ splenocytes, either isolated from 5-9 weeks old transgenic mice ortheir corresponding wt littermates were TCR-stimulated with α-CD3ε (10.0μg/ml) and increases in free intracellular Ca²⁺ were measured in realtime using a FACSAria flow-cytometer. The Ca²⁺-concentration of the usedRPMI-media was 2.0 mM. Data are representative of five independentexperiments with similar results.

FIG. 50: Calcium responses in CD4⁺ splenic T cells isolated fromtransgenic mice and their corresponding wt littermates. MACS®-purifiedCD4⁺ splenocytes, either isolated from 5-9 weeks old transgenic mice ortheir corresponding wt littermates were stimulated with both α-CD3ε(10.0 μg/ml) and α-CD28 (5.0 μg/ml). Increases in free intracellularCa²⁺ were measured in real time using a FACSAria flow-cytometer. TheCa²⁺-concentration of the used RPMI-media was 2.0 mM. Data arerepresentative of five independent experiments with similar results.

FIG. 51: MZB1 expression in MOCK-transduced and MZB1-transduced Fo Bcells. FACS-sorted FO B cells (B220⁺CD21^(int)CD23^(hi)) were eitherretrovirally transduced with GFP alone (MOCK) or with MZB1 and GFP(+MZB1). Transduced, GFP⁺ Fo B cells were enriched by FACS-sorting. Animmunoblot analysis with MZB1-specific antibodies (clone 2F9) wasperformed on 20 mg total protein extracts from splenic FO B cells,infected with the MZB1- and GFP-expressing (+MZB1) or the solelyGFP-expressing retrovirus (MOCK). As a control for loading and transferan additional immunoblot analysis using a GAPDH-specific antibody wasperformed.

FIG. 52: Measurement of [³H]thymidine incorporation in MOCK-transducedand MZB1-transduced FO B cells. FACS-sorted FO B cells(B220⁺CD21^(int)CD23^(hi)) were either retrovirally transduced with GFPalone (MOCK) or with MZB1 and GFP (+MZB1). Transduced, GFP⁺ FO B cellswere enriched by FACS-sorting. The infected and FACS-sorted (GFP⁺) FO Bcells were plated in a 96 well format. Each well was pulsed with 1.5 mCi[³H] thymidine 12 h-16 h before being collected. Data are expressed asthe mean [³H] thymidine incorporation of triplicate cultures. The figureis a representative of three independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors identified a novel gene from mouse B cells, whichis expressed at high levels in splenic MZ B cells and peritoneal B1 Bcells, but is expressed at very low level or not expressed at all insplenic follicular (FO) B cells. The gene encodes a protein, which hasan apparent molecular weight of 21 kD on SDS gel, which the inventorsnamed “MZB1” (marginal zone B1 B cell factor 1). The present inventorsfound that MZB1 acts as a modulator of the BCR signal transductioncascade in MZ and B1 B cells, possibly via its interaction with RACK1and PKCβ. Specifically, MZB1 negatively regulates B cell proliferationand tyrosine phosphorylation in the absence of BCR stimulation andcalcium mobilization upon BCR stimulation. Furthermore, the presentinventors found that MZB1 is a positive regulator of antibody productionfrom B cells, not only MZ and B1 B cells, but also FO B cells, possiblyvia its interaction with BiP.

As used herein, “a” and “an” refer to not only a single individual, butalso a group or species of entities.

All terms used herein bear the meanings that are established in the artunless otherwise noted. All techniques disclosed herein can be performedby a person skilled in the art following established protocols, such asthose disclosed in Molecular Cloning: A Laboratory Manual (Sambrook etal., 1989, Cold Spring Harbour Laboratory, New York), Current Protocolsin Molecular Biology (Ausubel et al., 2007, John Wiley & Sons, NewYork), Current Protocols in Immunology (Coligan et al., 2007, John Wiley& Sons, New York), Current Protocols in Protein Science (Coligan et al.,2007, John Wiley & Sons, New York) and Antibodies, a Laboratory Manual(Harlow et al., 1988, Cold Spring Harbour Laboratory, New York).

Nucleic Acid Molecules

The present invention provides an isolated nucleic acid moleculecomprising or having a nucleotide sequence encoding a polypeptide havingat least one of the biological activities of MZB1.

As used herein, the term “isolated nucleic acid” refers to a nucleicacid that is removed from its natural environment such as genomic DNAand that is free from at least one contaminating nucleic acid, proteinor other substances with which it is naturally associated, andpreferably substantially free from any contaminating nucleic acid,protein or other substances which would interfere with its use inprotein production or other uses.

As disclosed herein, MZB1 positively regulates antibody production,negatively regulates proliferation and tyrosine phosphorylation in theabsence of BCR stimulation and Ca²⁺ mobilization in the presence of BCRstimulation in a B cell, such as a MZ B cell, a B1 B cell and a FO Bcell.

In a preferred embodiment, MZB1 is encoded by the nucleotide sequence ofSEQ ID NO:1, 3, 11, 12 or 13, or a nucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO: 2 or 4,orthologs, allelic variants, or splice variants thereof. A nucleic acidmolecule comprising or having any of the above-mentioned nucleotidesequence is hereafter referred to “an MZB1 nucleic acid molecule”.

An ortholog refers to a gene in a different species that has evolvedfrom a common ancestor as the gene comprising the nucleotide sequence ofSEQ ID NO:1 or 3 or the nucleotide sequence encoding a polypeptidehaving the amino acid sequence of SEQ ID NO: 2 or 4.

An allelic variant refers one of several possible naturally occurringalternate forms of the gene comprising the nucleotide sequence of SEQ IDNO:1 or 3 or the nucleotide sequence encoding a polypeptide having theamino acid sequence of SEQ ID NO: 2 or 4 which occupy a given locus on achromosome of an organism or a population of organisms.

A splice variant refers an mRNA which is generated by alternativesplicing of an RNA transcript from the gene comprising the nucleotidesequence of SEQ ID NO:1 or 3 or the nucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO: 2 or 4.

In another preferred embodiment, MZB1 has the amino acid sequence of SEQID NO:2 or 4. In yet another preferred embodiment, MZB1 has the aminoacid sequence of SEQ ID NO:2 or 4 further comprising an amino-terminalmethionine.

The amino acid sequence of SEQ ID NO:2, encoded by the nucleotidesequence of SEQ ID NO:1, represents the sequence of full length MZB1including the signal peptide. The amino acid sequence of SEQ ID NO:4,encoded by the nucleotide sequence of SEQ ID NO:3, represents thesequence of mature MZB1 lacking the signal peptide.

The isolated nucleic acid molecule of the present invention includes aMZB1 nucleic acid molecule which comprises or has a nucleotide sequenceselected from the group consisting of:

-   (a) a nucleotide sequence as set forth in SEQ ID NO:1, 3, 11, 12 or    13,-   (b) a nucleotide sequence encoding the polypeptide having an amino    acid sequence as set forth in SEQ ID NO:2 or 4,-   (c) a nucleotide sequence encoding a polypeptide having the amino    acid sequence as set forth in SEQ ID NO:2 or 4 further comprising an    amino-terminal methionine,-   (d) a nucleotide sequence that is an ortholog of any of (a)-(c),-   (e) a nucleotide sequence that is an allelic variant or a splice    variant of any of (a)-(d).

The isolated nucleic acid molecule of the present invention alsoincludes a variant of the MZB1 nucleic acid molecules described above.

In one embodiment, the variant comprises or has a nucleotide sequencethat is at least 50%, 60%, 70%, 80%, 85%, preferably at least 90%, 91%,92%, 93%, 94%, more preferably at least 95%, 96%, 97%, 98%, even morepreferably at least 99% identical to the nucleotide sequence of any of(a)-(e) above, in particular, (a)-(c) above, over its entire length.

In another embodiment, the variant comprises or has a nucleotidesequence encoding a polypeptide that has at least 72%, 75%, 80%, 85%,preferably at least 90%, 91%, 92%, 93%, 94%, more preferably at least95%, 96%, 97%, 98%, even more preferably at least 99% amino acidsequence identity or similarity to the polypeptide as set forth in SEQID NO:2 or 4, orthologs, allelic variants or splice variants thereofover its entire length.

The term “identity” has well-established meaning in the art and refersto the percent of identical matches between two or more sequences withgap alignments addressed by a particular mathematical model of computerprograms (i.e., “algorithms”).

The term “similarity” has well-established meaning in the art and refersto a measure of similarity which includes both identical matches andconservative substitution matches between two or more sequences with gapalignments addressed by a particular mathematical model of computerprograms (i.e., “algorithms”). Since conservative substitutions apply topolypeptides and not nucleic acid molecules, similarity only deals withpolypeptide sequence comparisons.

The term “conservative amino acid substitution” has well-establishedmeaning in the art and refers to a substitution of a native amino acidresidue with a non-native residue such that there is little or no effecton the size, polarity or charge of the amino acid residue at thatposition. A person skilled in the art knows which amino acidsubstitutions are conservative and which are not.

Identity and similarity between nucleic acid molecules and polypeptidescan be readily calculated by known methods. Preferred methods todetermine identity and/or similarity are designed to give the largestmatch between the sequences tested. Methods for determining identity andsimilarity are codified in publicly available computer programs.

Preferably, the degree of sequence identity and/or similarity betweentwo sequences is determined by the BLAST 2 SEQUENCES publicly availableon the NCBI website(http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) using the defaultsetting. The algorithm employed is based on Tatiana A, et al. (1999).

The default setting of version BLASTN 2.2.17 (updated Aug. 26, 2007) forcomparing two nucleotide sequences includes:

-   -   reward for match: 1    -   penalty for a mismatch: 2    -   penalty for open gap: 5    -   penalty for gap extension: 2

The default setting of version BLASTP2.2.17 (updated Aug. 26, 2007) forcomparing two amino acid sequences includes:

-   -   matrix: BLOSUM62    -   penalty for open gap: 11    -   penalty for gap extension: 1

The term “over its entire length” means that the degree of identity andsimilarity is expressed as a percentage of the number of identical oridentical plus similar residues over the total number of residues of thereference sequence. Therefore, “sequence B is 50% identical to sequenceA over its entire length” means that “sequence B is 50% identical tosequence A over the entire length of sequence A”. Similarly, “sequence Bhas 50% identity (or similarity) to sequence A over its entire length”means that “sequence B has 50% sequence identity (or similarity) overthe entire length of sequence A”. For example, if sequence A has 100residues, sequence B has 75 residues, sequence A and B have 50 identicalresidues in an overlap of 60 residues, then sequence B is 50/100=50%identical to sequence A.

In yet another embodiment, the variant comprises or has a nucleotidesequence which hybridizes under highly stringent or moderately stringentconditions to the nucleotide sequence of any of (a)-(e) above, inparticular, (a)-(c) above, or a complementary sequence thereof.

The conditions of high and moderate stringency are well known to thoseskilled in the art and can be found in standard protocol books such asMolecular Cloning: A Laboratory Manual (Sambrook et al., supra) andCurrent Protocols in Molecular Biology (Ausubel et al., supra).

In a further embodiment, the variant comprises or has a nucleotidesequence encoding a polypeptide having the amino acid sequence encodedby a MZB1 nucleic acid molecule, in particular, the amino acid sequenceof SEQ ID NO:2 or 4, with at least one modification, preferably 1-150modification(s), more preferably 1-100 modification(s), even morepreferably 1-50 modification(s), most preferably 1-30 modification(s),selected from the group consisting of amino acid substitution, aminoacid insertion, amino acid deletion, carboxyl-terminal truncation, andamino-terminal truncation.

In particular, the variant comprises or has a nucleotide sequenceencoding a polypeptide having the amino acid sequence encoded by a MZB1nucleic acid molecule, in particular, the amino acid sequence of SEQ IDNO:2 or 4, with 1, 2, 3, 4, 5, 1-10, 1-15, 1-20 or 1-30 amino acidsubstitution(s), 1, 2, 3, 4, 5, 1-10, 1-15, 1-20 or 1-30 amino acidinsertion(s), 1, 2, 3, 4, 5, 1-10, 1-15, 1-20 or 1-30 amino aciddeletion(s), a carboxyl-terminal truncation of 1, 2, 3, 4, 5, 1-10,1-15, 1-18, 1-20, 1-30, 1-50, 1-100 or 1-150 amino acid(s), anamino-terminal truncation of 1, 2, 3, 4, 5, 1-10, 1-15, 1-18, 1-20,1-30, 1-50, 1-100 or 1-150 amino acid(s), or any combination thereof.

In certain specific embodiments, the variant comprises or has anucleotide sequence as set forth in SEQ ID NO: 5, 7 or 9. In otherspecific embodiments, the variant comprises or has a nucleotide sequenceencoding a polypeptide having an amino acid sequence as set forth in SEQID NO: 6, 8 or 10.

The term “amino acid substitution” refers to both conservative andnon-conservative amino acid substitutions. Amino acid substitution alsoencompasses substitution with non-naturally occurring amino acidresidues such as those typically incorporated by chemical peptidesynthesis. Non-naturally occurring amino acid residues includepeptidomimetics, and other reversed or inverted forms of amino acidmoieties.

In one special embodiment, the variant encodes a dominant-negativevariant of MZB1 which inhibits at least one of the biological activitiesof MZB1.

The present invention also provides a fragment of a MZB1 nucleic acidmolecule or a variant thereof described above.

In one embodiment, the fragment comprises or contains at least 12, 15,18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 45, 60, 75, 90, 120, 150, 180,210, 240, 270, 300, 360, 450, or 540 consecutive nucleotides of a MZB1nucleic acid molecule or a variant thereof, in particular, a nucleicacid molecule comprising or having the nucleotide sequence of SEQ IDNO:1 or 2 or encoding the amino acid sequence of SEQ ID NO:2 or 4.

In another embodiment, the fragment comprises or has a nucleotidesequence encoding a polypeptide of at least 4, 5, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 120, 150, or 180 consecutive amino acidresidues of any of the polypeptides encoded by a MZB1 nucleic acidmolecule or a variant thereof, in particular, a polypeptide encoded bythe nucleotide sequence of SEQ ID NO:1 or 2 or having the amino acidsequence of SEQ ID NO:2 or 4.

The nucleic acid fragment of the present invention may encode a peptideor polypeptide which has at least one of the biological activities ofMZB1. The nucleic acid fragment of the present invention may encode apeptide or polypeptide which is useful as a research tool, such as animmunogen, or a bait for interaction partner(s). The nucleic acidfragment of the present invention may be used in the detection ormodulation (i.e., increase or decrease) of expression of a MZB1 nucleicacid molecule or a variant thereof.

The present invention further provides a nucleic acid moleculecomprising or having a nucleotide sequence that is complementary to thenucleotide sequence of any of the nucleic acid molecules describedabove.

The degree of complementarity is preferably at least 50%, 60%, 70%, morepreferably at least 75%, 80%, 85%, 90%, even more preferably at least95%, 96%, 97%, 98%, 99%, and most preferably 100%.

As used in the art, the term “degree of complementarity” between twooligonucleotides or polynucleotides refers to the percentage ofcomplementary bases in the overlapping region of the twooligonucleotides or polynucleotides. Two bases are complementary to eachother if they can form a base pair via hydrogen bonding. Base pairsinclude both Waston-Crick base pairs (A-T, C-G, A-U) and wobble basepairs (G-U, I-U, I-A, I-C). The degree of complementarily can bedetermined by a skilled person using any known methods in the art. Forexample, ATCG has 100% complementarity to CGAT and CGATGG, and 75%complementarity to CGTT and CGTTGG.

In one embodiment, the complementary nucleic acid molecule is at least12, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 40 or 50 nucleotides inlength.

In another embodiment, the complementary nucleic acid comprises one ormore stretches of non-complementary sequence(s).

The complementary nucleic acid molecule may be used in the detection ormodulation (i.e., increase or decrease) of expression of a MZB1 nucleicacid molecule or a variant thereof.

In a preferred embodiment, the complementary nucleic acid molecule iscapable of downregulating the expression of a polypeptide encoded by aMZB1 nucleic acid molecule, a variant or a fragment thereof.

In certain embodiments, the complementary nucleic acid molecule is anantisense RNA, an siRNA, an shRNA, a miRNA, or a ribozyme. A personskilled in the art knows very well the meaning of the above terms andthe structural requirements and the functions of the above entities.

The present invention additionally provides a nucleic acid moleculewhich encodes a fusion polypeptide comprising a MZB1 polypeptide, avariant or a fragment thereof as described below fused to a heterologousamino acid sequence.

The isolated nucleic acid molecule of the present invention may besingle-stranded, single-stranded with a hairpin structure,double-stranded, or partially double-stranded.

The isolated nucleic acid molecule of the present invention may be DNA,RNA, or chimeric or hybrid DNA-RNA. The nucleic acid of the presentinvention may contain naturally occurring nucleotides, modifiednucleotides, or any of the known base analogs of DNA and RNA.Furthermore, the nucleic acid of the present invention may be modifiedcovalently or non-covalently.

In a preferred embodiment, the nucleic acid of the present invention, inparticular, an RNA, is modified to have enhanced chemical stabilityand/or nuclease resistance. Exemplary modifications include theintroduction of phosphorothioate linkage(s) and/or pyrophosphatelinkage(s).

The isolated nucleic acid molecules of the present invention can beobtained by a person skilled in the art using techniques established inthe art, such as library screening, PCR amplification, in vitrotranscription, and chemical synthesis.

The isolated nucleic acid molecules of the present invention may be usedto modulate (i.e., increase or decrease) the expression level of theencoded polypeptide in a cell or an organism which expresses thepolypeptide endogenously or a host cell or a host organism whichexpresses the polypeptide exogenously. The isolated nucleic acidmolecules of the present invention may also be used in detection anddiagnosis.

Polypeptides

The present invention provides an isolated polypeptide having at leastone of the biological activities of MZB1.

The term “isolated polypeptide” refers to a polypeptide this is removedfrom its natural environment such as a cell and that is free from atleast one contaminating polypeptide or other contaminating substancethat is found in its natural environment, and preferably substantiallyfree from any contaminating polypeptides and contaminating substanceswhich would interfere with its use.

The isolated polypeptide of the present invention includes a MZB1polypeptide which comprises or has the amino acid sequence of SEQ IDNO:2 or 4 or the amino acid sequence of SEQ ID NO:2 or 4 furthercomprising an amino-terminal methionine, an ortholog, allelic variant orsplice variant thereof, or which is encoded by a MZB1 nucleic acid ofthe present invention.

The polypeptide of the present invention also includes a variant of aMZB1 polypeptide.

In one embodiment, the variant is encoded by a nucleotide sequence thatis at least 50%, 60%, 70%, 80%, 85%, preferably at least 90%, 91%, 92%,93%, 94%, more preferably at least 95%, 96%, 97%, 98%, even morepreferably at least 99% identical to the nucleotide sequence of a MZB1nucleic acid molecule, in particular, the nucleotide sequence of SEQ IDNO:1 or 3, over its entire length.

In another embodiment, the variant has at least 72%, 75%, 80%, 85%,preferably at least 90%, 91%, 92%, 93%, 94%, more preferably at least95%, 96%, 97%, 98%, even more preferably at least 99% amino acidsequence identity or similarity to a MZB1 polypeptide, in particular,the polypeptide comprising or having the amino acid sequence of SEQ IDNO:2 or 4 over its entire length, optionally further comprising anamino-terminal methionine.

In a further embodiment, the variant is encoded by a nucleic acidcomprising or having a nucleotide sequence which hybridizes under highlystringent or moderately stringent conditions to a MZB1 nucleic acidmolecule or a complementary molecule thereof, in particular, thenucleotide sequence as set forth in SEQ ID NO:1 or 3, optionally furthercomprising an amino-terminal methionine.

In a still further embodiment, the variant comprises or has an aminoacid sequence of a MZB1 polypeptide, in particular, the amino acidsequence as set forth in SEQ ID NO:2 or 4, with at least onemodification, preferably 1-150 modification(s), more preferably 1-100modification(s), even more preferably 1-50 modification(s), mostpreferably 1-30 modification(s) selected from the group consisting ofamino acid substitution, amino acid insertion, amino acid deletion,carboxyl-terminal truncation, and amino-terminal truncation, optionallyfurther comprising an amino-terminal methionine.

In particular, the variant comprises or has an amino acid sequence of aMZB1 polypeptide, in particular, the amino acid sequence as set forth inSEQ ID NO:2, with 1, 2, 3, 4, 5, 1-10, 1-15, 1-20 or 1-30 amino acidsubstitution(s), 1, 2, 3, 4, 5, 1-10, 1-15, 1-20 or 1-30 amino acidinsertion(s), 1, 2, 3, 4, 5, 1-10, 1-15, 1-20 or 1-30 amino aciddeletion(s), a carboxyl-terminal truncation of 1, 2, 3, 4, 5, 1-10,1-15, 1-18, 1-20, 1-30, 1-50, 1-100 or 1-150 amino acid(s), anamino-terminal truncation of 1, 2, 3, 4, 5, 1-10, 1-15, 1-18, 1-20,1-30, 1-50, 1-100 or 1-150 amino acid(s), or any combination thereof,optionally further comprising an amino-terminal methionine.

In certain specific embodiments, the variant is encoded by a nucleotidesequence as set forth in SEQ ID NO: 5, 7 or 9. In other specificembodiments, the variant comprises or has an amino acid sequence as setforth in SEQ ID NO: 6, 8 or 10.

In one special embodiment, the variant is a dominant-negative variant ofMZB1 which inhibits at least one of the biological activities of MZB1.

The present invention also provides a fragment of a MZB1 polypeptide ora variant thereof described above.

In one embodiment, the fragment is encoded by a nucleic acid moleculecomprising or having at least 12, 15, 18, 21, 24, 30, 45, 60, 75, 90,120, 150, 180, 210, 240, 270, 300, 360, 450, 540 consecutive nucleotidesof a nucleic acid molecule of the present invention, preferably, thenucleic acid molecule having the nucleotide sequence as set forth in SEQID NO:1 or 3, optionally further comprising an amino-terminalmethionine.

In another embodiment, the fragment comprises or consists of at least 5,10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 180consecutive amino acid residues of a MZB1 polypeptide or a variantthereof, preferably, the polypeptide comprising or having the amino acidsequence of SEQ ID NO:2 or 4, optionally further comprising anN-terminal methionine.

The peptide or polypeptide fragment may have at least one of thebiological activities of MZB1. The peptide or polypeptide fragment maybe useful as a research tool, such as an immunogen, or a bait forinteraction partner(s).

The present invention further provides a fusion polypeptide comprising aMZB1 polypeptide, a variant or a fragment thereof described above fusedto a heterologous amino acid sequence.

The heterologous amino acid sequence may be an epitope tag which may beuseful in the purification and/or the detection of the fusionpolypeptide. Commonly used epitope tags include, but are not limited to,His tag (poly-His or hexa-His), Myc tag, FLAG tag, and HA tag.

The heterologous amino acid sequence may be Fc domain of animmunoglobulin (i.e., antibody) molecule or any other sequence which maybe useful in the purification and the detection of the fusionpolypeptide.

The heterologous amino acid sequence may be sequences that are involvedin the dimerization or multimerization of the fusion protein, including,but not limited to, the leucine zipper sequences and the Fc domain of anantibody.

The heterologous amino acid sequences may also be sequences that arecapable of targeting the fusion protein to particular tissues, cells,subcellular compartments, or facilitating the transport of the fusionprotein across the cell membrane.

The polypeptide of the present invention may be chemically modified, forexample, by covalent attachment of one or more polymers. Themodification may alter the biological activity, chemical stability,bioavailability, biodistribution, pharmacokinetic properties of thepolypeptide.

The polypeptide of the present invention may be obtained from naturallyoccurring cells, such as MZ B cells and B1 B cells, which express thepolypeptide endogenously, or host cells, host organisms, or parts ofhost organisms which express the polypeptide exogenously.

Polypeptides, in particular, fragments of the present invention may alsobe prepared by chemical synthesis methods (such as solid phase peptidesynthesis) using techniques known in the art. The polypeptide may besynthesized with or without a methionine at the amino terminus.

The polypeptides of the present invention may be used as a researchtool, for example, to identify interaction partners. The polypeptide ofthe present invention may be used to screen for compounds which modulate(i.e., increase or decrease) at least one of the biological activitiesof MZB1. The polypeptide of the present invention may also be used as animmunogen for generating specific antibodies.

TABLE 1 MZB1 and selected variants and fragments. SEQ ID NO: nucleotideamino acid MZB1 polypeptide sequence sequence full length (aa1-188) 1 2mature (aa19-188) 3 4 full length without REEL (aa1-184) 5 6 maturewithout REEL (aa19-184) 7 8 fragment (aa46-179) 9 10 MZB1 nucleic acidfull length with 5′ and 3′ UTR 11 full length with 5′ UTR 12 full lengthwith 3′ UTR 13

Vectors

The present invention provides a vector comprising a nucleic acidmolecule of the present invention, operably linked to transcriptionregulatory sequences.

In one embodiment, the transcription regulatory sequences areheterologous transcription regulatory sequences which are different fromthe native transcription regulatory sequences for the MZB1 polypeptide.

The term “transcription regulatory sequences” refers to elements thatare invovled in the regulation (positive or negative) of transcription,including but not limited to, promoters and enhancers.

The term “operably linked” refers to an arrangement which allows for thetranscription and/or transcription and translation of the nucleic acidmolecule of the present invention.

Elements which are necessary for the maintenance, replication andselection of the vector, and the transcription and/or translation of theinserted nucleic acid sequence are well known in the art. Furthermore,elements which may enhance the transcription and/or translation of theinserted nucleic acid sequence are also well known in the art.

In one embodiment, the expression from the expression vector isconstitutive in a host cell, or constitutive and/or ubiquitous in a hostorganism. In another embodiment, the expression from the expressionvector is inducible in the host cell or host organism. In yet anotherembodiment, the expression from the expression vector is cell-, celltype-, cell lineage-, tissue-specific or developmental stage-specific inthe host organism.

The vector of the present invention allows for the transcription and/ortranscription and translation of the nucleic acid of the presentinvention. The vector of the present invention may be used to modulate(i.e., increase or decrease) the expression level of a polypeptide ofthe present invention in a cell or an organism which expresses thepolypeptide endogenously or in a host cell or a host organism whichexpresses the polypeptide exogenously.

A person skilled in the art can select an appropriate vector on thebasis of the host cell or host organism to be used and the purpose ofthe transcription of the nucleic acid of the present invention.

Host Cells

The present invention provides a host cell comprising a nucleic acid ora vector of the present invention.

The nucleic acid or the vector may exist extra-chromosomally or may beintegrated into the genome of the host cell.

The host cell of the present invention may be a prokaryotic cell or aeukaryotic cell. Prokaryotic host cells include, but are not limited to,bacterial cells such as various strains of E. coli, B. subtilis,Pseudomonas spp., Bacillus spp., Streptomyces spp. Eukaryotic host cellsinclude, but are not limited to, yeast, plant cells, invertebrate cellsand vertebrate cells. Invertebrate cells include insect cells;vertebrate cells include mammalian cells. Preferred yeast cells include,but are not limited to, Saccharomyces cerivisae, Schizosaccharomycespombe, and piccia. Preferred mammalian cells include, but are notlimited to, CHO, COS, 3T3, HeLa, 293, K46, EL4, WEHI231, WEHI279, BJAB,Raji, J558L, IL-7-dependent pre-B cells, hybridoma and myeloma cells.

A host cell of the present invention may be obtained by introducing avector of the present invention into a host cell via any of the methodswell known in the art, including, but not limited to, transformation(e.g., heat shock, electroporation), transfection (e.g., calciumphosphate precipitation, electroporation, lipofection, the DEAE-dextranmethod), infection or transduction (e.g., by bacteriophage or viruses),and microinjection. The method will at least in part depend on the typeof host cell to be used.

In certain embodiments, the nucleic acid of the present inventioncontains codons which have been altered for optimal expression in agiven host cell.

The host cell of the present invention may be used to the produce apolypeptide of the present invention. Certain appropriate host cells mayalso be used for producing antibodies.

Host Organisms

The present invention provides a host organism comprising a nucleic acidor a vector of the present invention.

In a perferred embodiment, the nucleic acid or the vector is stablyintegrated into the genome of the host organism. In other words, thehost organism of the present invention is a transgenic organism.

As used herein, the term “organism” refers to a multi-cellular organism.The host organism of the present invention is preferably a vetebrateanimal. In one embodiment, the host organism is a laboratory animal,including but not limited to, mouse, rat, guinea pig, hamster, rabbit,dog, monkey, chimpanzee. In another embodiment, the host organsim is afarm animal, including but not limited to, cow, ox, sheep, goat, horse,pig. In yet another embodiment, the host animal is a non-human mammal,such as a rodent or a non-human primate. In a preferred embodiment, theorganism is mouse.

Since the nucleic acid and the vector of the present application mayincrease or decrease the expression of a polypeptide of the invention,the transgenic organism of the present invention may have increased ordecreased expression of the polypeptide of the present inventioncompared to a non-transgenic counterpart.

The expression from the nucleic acid or the vector of the presentapplication in the host organism may be constitutive, ubiquitous,inducible, and/or cell-, cell type-, cell lineage-, tissue-specific ordevelopmental stage-specific.

The host organism of the present invention may be used as a researchtool for studying the biology of MZB1 using gain-of-function orloss-of-function approaches.

In a specific embodiment, the host organism of the present invention isa BAC (bacterial artificial chromosome) transgenic animal. The BACtransgenic animal carries a transgene construct which comprises a BACwhich contains the complete MZB1 genomic locus with all regulatorysequences. The coding sequence of MZB1 in the transgenic construct maybe substituted by the coding sequence of a MZB1 variant or a reporterprotein (such as GFP). The MZB1 variant may be a polypeptide encoded bythe nucleotide sequence of SEQ ID NO: 3, 5, 7 or 9, or a polypeptidehaving the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10.

Knock-out and Knock-in Organisms

The present invention provides a knock-out or a conditional knock-outorgansim in which the endogenous gene encoding MZB1 is inactivated orconditionally inactivated.

The present invention also provides a knock-in organism wherein anexogenous gene, such as a reporter gene (e.g., GFP), is introduced intothe mzb1 genetic locus via homologous recombination. The timing andpattern of expression of the exogenous gene reflects that of endogenousMZB1.

Methods for generating knock-out, conditional knock-out, or knock-inorganism are well known in the art.

The preferred organism is a vetebrate animal, in particular, a non-humanmammal. More preferably, the organism is a laboratory animal or a farmanimal. Mostly preferably, the organism is a rodent, in particular, amouse.

The knock-out and conditional knock-out organism may be used to studythe biological functions of MZB1 and to test the effectiveness of agentswhich promotes or decreases MZB1 expression and/or activity. Theknock-in organism may be used to study the regulation of MZB1expression.

Antibodies

The present invention provides an antibody or a fragment thereof thatspecifically binds to a polypeptide of the present invention.

The antibody of the present invention may be polyclonal or monoclonal.The antibody of the present invention may be a chimeric antibody, ahumanized antibody or a fully human antibody. The antibody of thepresent invention may be a single chain antibody. The antibdoy of thepresent invention may be bi-specific.

The antibody fragment of the present invention may be an Fab fragment,an Fab′ fragment, an F(ab′)₂ fragment, an Fv fragment, or any otherfragment which retains the antigen binding specificity of the intactantibody.

In a preferred embodiment, the antibody of the present invention is aneutralizing or antagonist antibody which inhibits at least one of thebiological activities of MZB1 upon binding MZB1.

Whether an antibody inhibits at least one of the biological activitiesof MZB1 can be readily determined by functional assays, such as Ca²⁺mobilization assay, cell proliferation assay, and tyrosinephosphorylation assay described in the Examples.

In one embodiment, the neutralizing or antagonistic antibody interfereswith the interaction between MZB1 and at least one of its interactionpartners, such as RACK1, PKCβ, and BiP. In a specific embodiment, theantibody interferes with the interaction between MZB1 and RACK1. In aparticular embodiment, the antibody is 2F9 which interferes with theinteraction between MZB1 and RACK1.

Whether an antibody interferes with the interaction between MZB1 and oneor more of its interaction partners can be readily determined by assayswell-known in the art, such as co-immunoprecipitation, FRET, gelfiltration.

In another preferred embodiment, the antibody of the present inventionis an agnostic antibody which activates or enhances at least one of thebiological activities of MZB1 upon binding MZB1. Whether an antibodyactivates or enhances at least one of the biological activities of MZB1can be readily determined by functional assays, such as Ca²⁺mobilization assay, cell proliferation assay, and tyrosinephosphorylation assay described in the Examples.

In yet another preferred embodiment, the antibody of the presentinvention is an intracellular antibody, i.e., an intrabody.

In one embodiment, the intrabody is a single-chain Fv fragment (scFv)which contains the heavy and the light chain variable region of anantibody linked by a flexible linker. scFv is expressed intracellularlyand bind its intracellular target. scFv may inhibit the function of atarget protein by directly inhibiting an enzymatic activity, inhibitingprotein-protein interaction, or interfering with the post-translationalmodification or the transport of the target protein. The fusion of anintracellular localization signal, such as a nuclear localization signal(NLS) or an endoplasmic reticulum retention signal, to the scFv allowsfor the targeting of the scFv to a specific subcellular compartment.

In another embodiment, the intrabody is an antibody or anantigen-binding fragment thereof fused to an internalization signal,such as that found in HIV TAT.

The present invention also provides an isolated nucleic acid moleculewhich encodes an antibody of the present invention, and a hybridoma or ahost cell that produces an antibody of the present invention.

The antibody and the antigen-binding fragment thereof, their codingsequence, the hybridoma and the host cell of the present invention canbe obtained by well-established methods in the art, such as thosedescribed in the Current Protocols in Immunology (Coligan et al., supra)and Antibodies, a Laboratory Manual (Harlow et al., supra).

The antibody and the antigen-binding fragment thereof of the presentinvention may be used for the purification, detection and quantificationof a polypeptide of the present invention. Furthermore, the antibody andthe antigen-binding fragment thereof of the present invention may beused for studying and/or modulating (i.e., increase or decrease) thebiological activities of the polypeptides of the present invention forresearch and clinical purposes.

Inhibitors of MZB1 Expression or Activity

The present invention provides a substance that inhibits the expressionof MZB1. The present invention also provides a substance that inhibitsat least one of the biological activities of MZB1.

Inhibitors of the expression of MZB1 include, but are not limited to,antisense RNA, RNA olignucleotides that are active in RNA interference(RNAi) such as siRNA, shRNA and miRNA, ribozyme, RNA and DNA aptamers,and decoy RNAs.

Given the coding sequence for MZB1, a person skilled in art can designand/or obtain antisense RNA, siRNA, shRNA, miRNA, decoy RNA, DNA and RNAaptamers using methods known in the art. For example, siRNA and shRNAmay be designed using publicly available algorithms such as thatdisclosed in Reynolds et al. (2004) and design engines such as “BD-RNAidesign” (Beckton Dickinson) and “Block-iT RNAi” (Invitrogen).Furthermore, a person skilled in the art can test the efficacy of theseagents in inhibiting MZB1 expression using methods known in the art suchas Northern blot analysis, quantitative or semi-quantitative RT-PCR,Western blot analysis, surface or intracellular FACS analysis.

In one embodiment, the inhibitor of MZB1 expression is an RNAoligonucleotide which is active in RNAi, such as an siRNA, shRNA andmiRNA, and which targets GCGAAAGCAGAGGCTAAAT (SEQ ID NO:14),GCAGTCCTATGGAGTTCAT (SEQ ID NO: 15) CCAGATCTATGAAGCCTAC (SEQ ID NO:16),or CTGCCACTGTTGCTACTGT (SEQ ID NO:17) within the MZB1 coding region. Incertain embodiments, the inhibitor of MZB1 expression is an siRNA. Incertain other embodiments, the inhibitor of MZB1 expression is an shRNA,in particular, an shRNA comprising the nucleotide sequence ofGCGAAAGCAGAGGCUAAAUUUCAAGAGAAUUUAGCCUCUGCUUUCGC (SEQ ID NO: 18),GCAGUCCUAUGGAGUUCAUUUCAAGAGAAUGAACUCCAUAGGACUGC (SEQ ID NO: 19),CCAGAUCUAUGAAGCCUACUUCAAGAGAGUAGGCUUCAUAGAUCUGG (SEQ ID NO: 20), andCUGCCACUGUUGCUACUGUUUCAAGAGAACAGUAGCAACAGUGGCAG (SEQ ID NO: 21) (thebold letters denotes the loop sequence and the normal letters denote theloop sequence).

Inhibitors of the activity of MZB1 include, but are not limited to,neutralizing antibodies, in particular, neutralizing intrabodies, RNAaptamer, DNA aptamers, peptide aptamers, small molecule inhibitors, adominant-negative MZB1, and a dominant-negative interaction partner.

Given the coding sequence of MZB1, a person skilled in the art canexpress and obtain the MZB1 protein using methods known in the art.Furthermore, a person skilled in the art can screen for agents whichinhibit at least one of the activities of MZB1 using either cell-basedor cell-free functional assays, such as Ca²⁺ mobilization assay, cellproliferation assay, tyrosine phosphorylation assay, antibody secretionassay described in the Examples.

For example, a person skilled in the art can contact MZB1-expressing andMZB1-deficient cells with a candidate agent, subject the cells to one ormore functional assays, and identify candidate agents which negativelyaffect at least one of the activities of MZB1 in MZB1-expressing but notMZB1-deficient cells. The MZB1-expressing and MZB1-deficient cellsshould be identical or essentially identical to each other except theexpression of MZB1.

In one example, the MZB1-deficient cells do not express any endogenousMZB1; the MZB1-expressing cells are their genetically modifiedcounterparts which express exogenous MZB1. In one specific example, theMZB1-expressing cells are MZB1-deficient cells transfected or transducedin vitro with an MZB1 expression vetor. In another specific example, theMZB1-expressing cells are isolated from an MZB1 transgenic animal andthe MZB1-deficient cells are isolated from its non-transgeniclittermate. In yet another specific example, the cells contain an MZB1expression vector under the control of an inducible promoter; byculturing cells under inducing and non-inducing conditions,MZB1-expressing and MZB1-deficient cells, respectivel, can be obtained.The MZB1 inducible cells can be obtained by in vitro transfection ortransduction or isolated from transgenic animals.

In another example, the MZB1-expressing cells express endogenous MZB1;the MZB1-deficient cells are their knock-out or knock-down counterpartswhich have no or reduced MZB1 expression. MZB1 knock-out cells may beobtained from an MZB1 knock-out host organism or via in vitro homologousrecombination. MZB1 knock-down cells may be obtained by the in vitrointroduction of an active RNAi agent (such as an siRNA, shRNA, ormiRNA), an antisense RNA, a ribozyme, an RNA aptamer, a DNA aptamer, ora decoy RNA or from a transgenic host organism which expresses one ormore of the above-mentioned agents. In addition, conditional knock-outor knock-down cells, either generated in vitro via transfection ortransduction or obtained from conditional knock-out or knock-downanimals, may be used to provide both MZB1-expressing and MZB1-deficientcells when grown under appropriate conditions.

An MZB1-specific inhibitor should inhibit at least one of the activitiesof MZB1 in MZB1-expressing cells, affect MZB1 knock-down cells to alesser degree and not affect MZB1 knock-out or non-expressing cells atall. MZB1 knock-down cells which different degrees of MZB1downregulation can serve as highly valuable controls for determining thespecificity of the candidate MZB1 activity inhibitor.

In a further example, a person skilled in the art can contact cellsexpressing wild-type (wt) MZB1 and cells expressing a mutant MZB1 with acandidate agent, subject the cells to one or more functional assays, andidentify candidate agents which negatively affect at least one of theactivities of MZB1 in cells expressing wt but not mutant MZB1.Experimental observations suggest that the ability of MZB1 to regulateantibody production is attributed to the pool of MZB1 which isassociated with the endoplasmic reticulum (ER) and the ability of MZB1to regulate cell proliferation is attributed to the pool of MZB1 that isin the cytoplasm and can be recruited to the plasma membrane. Withoutwishing to be bound by any theory, cells expressing MZB1 mutants whichhave altered subcellular localization may serve as controls fordetermining the specificity of a candidate inhibitor which inhibits oneor more subcellular localization-associated function(s) of MZB1. In oneexample, cells expressing an MZB1 mutant lacking the N-terminal signalpeptide (i.e., the N-terminal 18 amino acids; e.g., a polypeptide havingthe amino acid sequence of SEQ ID NO: 4 or encoded by the nucleotidesequence of SEQ ID NO:3) can be used as a control in the identificationof agents which inhibit at least one of the cytoplasmic functions ofMZB1, such as the regulation of cell proliferation. In another example,cells expressing an MZB1 mutant lacking the C-terminal ER retentionsignal (i.e., the C-terminal 4 amino acids REEL; e.g., a polypeptidehaving the amino acid sequence of SEQ ID NO: 6 or 8 or encoded by thenucleotide sequence of SEQ ID NO: 5 or 7) can be used as a control inthe identification of agents which inhibit at least one of theER-associated functions of MZB1, such as antibody secretion.

An inhibitor of MZB1 activity may interfere with the interaction betweenMZB1 and one or more of its interaction partners. Whether a substanceinterferes with the binding of MZB1 to one or more of its interactionpartners can be determined by assays well known in the art, such asco-immunoprecipitation, FRET, gel filtration.

An inhibitor of MZB1 activity can be obtained by a person skilled in theart by screening candidate agents, such a panel of antibodies, a phagedisplay library of single-chain antibodies, an RNA aptamer library, aDNA aptamer library, a peptide aptamer library, a small moleculelibrary, a panel of potential dominant-negative MZB1 variants, and apanel of potential dominant-negative mutants of interaction partners,using one or more activity (i.e., functional) and/or binding assays.

Since MZB1 interacts with multiple interaction partners and has multiplebiological activities, it may be preferable to obtain an inhibitor ofMZB1 activity which inhibits only one or more, but not all, of thebiological activities of MZB1.

In one embodiment, an activity-specific inhibitor interferes with thebinding of MZB1 with only one or more, but not all, interactionpartner(s). In another embodiment, an activity-specific inhibit bindsand interferes with the function of only one or more, but not all,functional domains of MZB1. In yet another embodiment, anactivity-specific inhibitor is targeted to a specific subcellularcompartment and inhibits one or more subcellular localization-specificfunction(s) of MZB1. For example, an intrabody with an ER retentionsignal may be able to inhibit ER-associated function(s) of MZB1 such asthe regulation of antibody secretion without affecting the cytoplasmicfunction(s) of MZB1 such as the regulation of cell proliferation.

The inhibitor of MZB1 expression or activity may comprise anymodifications that improve its stability, bioavailability, and/orpharmacokinetic properties and/or modify its biodistribution and/orsubcellular localization.

The inhibitors of MZB1 expression or activity may be used for researchas well as medical purposes.

Activators or Enhancers of MZB1 Expression or Activity

The present invention provides a substance that activates or enhancesMZB1 expression. The present invention also provides a substance thatactivates MZB1 or enhances at least one of the biological activities ofMZB1.

Activators or enhancers of MZB1 expression include, but are not limitedto, nucleic acid molecules or vectors which are capable of driving theexpression of MZB1, a variant or a biologically active fragment thereof,and factors, such as transcription factors, which activate or enhancethe transcription from an MZB1 coding sequence.

Examples of vectors which are capable of driving the expression of MZB1,a variant or a biologically active fragment therefore include theretroviral vector pEG2-MZB1, and the MZB1-expression vectorspMZB1_(AUG2) driven by the CMV promoter, pCG-FLAG-MZB1 (N-terminalFLAG-tag) and pCG-MZB1-HA (C-terminal HA-tag).

Transcription factors, such as XBP-1, Blimp-1, IRF4, and Pax5 which areimportant players in the regulation of plasma cell differentiation andantibody secretion, are predicted to bind to the putative MZB1 promoterand thereby regulate the expression of endogenous MZB1.

Furthermore, MZB1 expression can be induced by factors such as bacterialcomponents like LPS.

Given the genomic sequence of the mzb1 locus, a person skilled in theart can identify factors which activate or enhance the transcriptionfrom the mzb1 gene using methods known in the art.

Activators or enhancers of MZB1 activity include, but are not limitedto, MZB1, a variant or a biologically active fragment thereof, agonisticantibodies, in particular, agonistic intrabodies, and small moleculeagonists.

Given the coding sequence of MZB1, a person skilled in the art canexpress and obtain the MZB1 protein using methods known in the art.Furthermore, a person skilled in the art can screen for agents whichactivate MZB1 or enhance at least one of the activities of MZB1 usingeither cell-based or cell-free assays, such as those described in theExamples. The same assays and strategies for identifying inhibitors ofMZB1 activity can be used for identifying activators or enhancers ofMZB1 activity.

Since MZB1 interacts with multiple interaction partners and has multiplebiological activities, it may be preferable to generate and utilize avariant or a fragment of MZB1 which has only one or more, but not all,of the biological activities of full length MZB1. Furthermore, sinceMZB1 in different subcellular compartments appears to carry outdifferent biological activities, it may be preferable to generate andutilize a variant or a fragment of MZB1 with a desired subcellularlocalization. Examples of such variants or fragments include thepolypeptides having the amino acid sequence of SEQ ID NOS: 4, 6, 8, 10or the polypeptides encoded by the nucleotide sequence of SEQ ID NOS: 3,5, 7, 9.

Moreover, it may be preferable to obtain an antibody or small moleculeagonist which activates or enhances only one or more, but not all, ofthe activities of MZB1.

In one embodiment, an activity-specific activator or enhancer interactswith only one or more, but not all, functional domains of MZB1. Inanother embodiment, an activity-specific activator or enhancer istargeted to a specific subcellular compartment and activates or enhancesone or more subcellular localization-specific function(s) of MZB1. Forexample, an intrabody with an ER retention signal may be able toactivates or enhances ER-associated function(s) of MZB1 such as theregulation of antibody secretion without affecting the cytoplasmicfunction(s) of MZB1 such as the regulation of cell proliferation.

The activator or enhancer of MZB1 expression or activity may compriseany modifications that improve its stability, bioavailability, and/orpharmacokinetic properties and/or modify its biodistribution and/orsubcellular localization.

The activators or enhancers of MZB1 expression or activity may be usedfor research as well as medical purposes.

Pharmaceutical Compositions

The present invention provides a pharmaceutical composition comprisingan isolated nucleic acid or a vector of the present invention and apharmaceutically acceptable carrier.

The present invention also provides a pharmaceutical compositioncomprising an isolated polypeptide of the present invention and apharmaceutically acceptable carrier.

The present invention further provides a pharmaceutical compositioncomprising an inhibitor of MZB1 expression or activity of the presentinvention and a pharmaceutically acceptable carrier.

The present invention additionally provides a pharmaceutical compositioncomprising an activator or enhancer of MZB1 expression or activity ofthe present invention and a pharmaceutically acceptable carrier.

In one embodiment, the active ingredient is modified to achieve improvedstability, bioavailability, pharmacokinetic properties, biodistribution(in particular, organ-, tissue-, or cell type-specificity), and/orsubcellular localization.

In one embodiment, the pharmaceutical composition further comprises anagent which facilitates the delivery of the active ingredient. Forexample, when the active ingredient is a DNA or RNA molecule, thepharmaceutical composition may further comprise a DNA or RNAcomplexation agent such as a liposome or a polycationic peptide.

In certain embodiments, the pharmaceutical composition of the presentinvention further comprises one or more additional pharmaceuticallyactive and/or therapeutic agents which are used for the treatment ofautoimmune disorders and immunodeficiency in a mammal.

The pharmaceutical composition of the present invention may beformulated in any way that is compatible with its therapeuticapplication, including intended route of administration, delivery formatand desired dosage. Optimal pharmaceutical compositions may beformulated by a skilled person according to common general knowledge inthe art, such as that described in Remington's Pharmaceutical Sciences(18th Ed., Gennaro AR ed., Mack Publishing Company, 1990).

The pharmaceutical compositions of the present invention may beformulated for instant release, controlled release, timed-release,sustained release, extended release, or continuous release.

The pharmaceutical compositions provided by the present invention may beadministered by any routes known in the art, including, but not limitedto, topical, enteral and parenteral routes. Topic administrationincludes, but is not limited to, epicutaneous, inhalational, intranasal,vaginal administration, enema, eye drops, and ear drops. Enteraladministration includes, but is not limited to, oral, rectaladministration and administration through feeding tubes. Parenteraladministration includes, but is not limited to, intravenous,intraarterial, intramuscular, intracardiac, subcutaneous, intraosseous,intradermal, intrathecal, intraperitoneal, transdermal, transmucosal,and inhalational administration.

The optimal dosage, frequency, timing and route of administration can bedetermined by a person skilled in the art.

The dosage regimen utilizing the inhibitor of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;and the particular compound employed. It will be acknowledged that anordinarily skilled physician or veterinarian can easily determine andprescribe the effective amount of the compound required to prevent,counter or arrest the progress of the condition.

Preferably, the pharmaceutical composition of the present invention issuitable for administration to a patient. In the context of the presentinvention the term “patient” means an individual in need of a treatmentof an autoimmune disease or immunodeficiency. Preferably, the patient isa vertebrate, even more preferred a mammal, particularly preferred ahuman.

The terms “treatment” and “treating” are used herein to generally meanobtaining a desired pharmaceutical and/or physiological effect.Preferably, the effect is therapeutic in terms of partially orcompletely curing an autoimmune disease or immunodeficiency. The term“treatment” as used herein covers any treatment of tissue suffering froman autoimmune disease or immunodeficiency and/or organ defects and/ordysfunction caused by an autoimmune disease or immunodeficiency in amammal, particularly a vertebrate and more preferably a human, andincludes regenerating and/or repairing suffering from an autoimmunedisease or immunodeficiency and/or organ or tissue dysfunction. Thus,the pharmaceutical composition of the present invention is preferablysuitable for the prevention and/or treatment of an autoimmune disease orimmunodeficiency.

The term “prevention” or “preventing” when used herein means to obtain aprotective effect on a tissue which is already suffering from anautoimmune disease or immunodeficiency so as to prevent further damageand/or a protective effect on a tissue which is at a risk of sufferingfrom an autoimmune disease or immunodeficiency.

Accordingly, the pharmaceutical composition of the present invention forthe purpose of treating and/or preventing an autoimmune disease orimmunodeficiency may preferably be administered to a subject who is at arisk of an autoimmune disease or immunodeficiency and/or who alreadysuffers from an autoimmune disease or immunodeficiency. Thus, thepharmaceutical composition of the present invention may preferably beadministered to a subject who is diagnosed to be at a risk of anautoimmune disease or immunodeficiency and/or who already suffers froman autoimmune disease or immunodeficiency.

The term “administered” means administration of a therapeuticallyeffective dose of a pharmaceutical composition of the present invention.Preferably, said therapeutically effective dose is administered to apatient who has tissue suffering from an autoimmune disease orimmunodeficiency. Particularly preferred said therapeutically effectivedose is administered to a patient suffering from organ defects and/ordysfunction caused by an autoimmune disease or immunodeficiency. By“therapeutically effective amount” is meant a dose that produces theeffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques. As is known in the art, adjustments forsystemic versus localized delivery, age, body weight, general health,sex, diet, time of administration, drug interaction and the severity ofthe condition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

The uses, methods and compositions of the present invention areapplicable to both human therapy and veterinary applications. Thecompounds described herein having the desired therapeutic activity maybe administered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt %. The agents may be administered alone or incombination with other treatments.

Use of an Inhibitor of MZB1 Expression or Activity

The present invention provides an inhibitor of MZB1 expression oractivity for treating autoimmune diseases in a mammal, in particular,autoimmune diseases that are caused by and/or associated withauto-antibodies.

The present invention also provides the use of an inhibitor of MZB1expression or activity for the preparation of a pharmaceuticalcomposition for treating autoimmune diseases in a mammal, in particular,autoimmune diseases that are caused by and/or associated withauto-antibodies.

Autoimmune diseases include, but are not limited to, diabetes mellitus,arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,automimmune thyroiditis, psoriasis, Sjogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, autoimmune uveitis, allergic encephalomyelitis, pure redcell anemia, idiopathic thrombocytopenia, Wegener's granulomatosis,chronic active hepatitis, lichen planus, Graves' disease, sarcoidosis,primary biliary cirrhosis, and interstitial lung fibrosis.

In one embodiment, the inhibitor is an inhibitor of MZB1 expression,including but not limited to antisense RNA, RNA olignucleotides that areactive in RNA interference (RNAi) such as siRNA, shRNA and miRNA,ribozyme, RNA and DNA aptamers, and decoy RNAs.

In a specific embodiment, the inhibitor is an RNA oligonucleotide whichis active in RNAi, such as an siRNA, shRNA and miRNA, and which targetsGCGAAAGCAGAGGCTAAAT (SEQ ID NO: 14), GCAGTCCTATGGAGTTCAT (SEQ ID NO:15), CCAGATCTATGAAGCCTAC (SEQ ID NO: 16), or CTGCCACTGTTGCTACTGT (SEQ IDNO: 17) within the MZB1 coding region. In certain embodiments, theinhibitor of MZB1 expression is an siRNA. In certain other embodiments,the inhibitor of MZB1 expression is an shRNA, in particular, an shRNAcomprising the nucleotide sequence ofGCGAAAGCAGAGGCUAAAUUUCAAGAGAAUUUAGCCUCUGCUUUCGC (SEQ ID NO: 18),GCAGUCCUAUGGAGUUCAUUUCAAGAGAAUGAACUCCAUAGGACUGC (SEQ ID NO: 19),CCAGAUCUAUGAAGCCUACUUCAAGAGAGUAGGCUUCAUAGAUCUGG (SEQ ID NO: 20), andCUGCCACUGUUGCUACUGUUUCAAGAGAACAGUAGCAACAGUGGCAG (SEQ ID NO: 21) (thebold letters denotes the loop sequence and the normal letters denote theloop sequence).

In another embodiment, the inhibitor is an inhibitor of MZB1 activity,inlucding but not limited to neutralizing antibodies, in particular,neutralizing intrabodies, RNA aptamers, DNA aptamers, peptide aptamers,small molecule inhibitors, dominant-negative MZB1 and dominant-negativeinteraction partner.

In a preferred embodiment, the inhibitor inhibits only the antibodyproduction-promoting activity of MZB1. In one embodiment, the inhibitorinterferes with the binding between MZB1 and Bip-1. In anotherembodiment, the inhibitor interacts with the domain of MZB1 which isinvolved in the regulation of antibody secretion and interferes with itsfunction. In yet another embodiment, the inhibitor is targeted to theER.

In another preferred embodiment, the inhibitor inhibits only theproliferation-inhibiting and/or BCR signaling-inhibiting activity ofMZB1. In one embodiment, the inhibitor interferes with the bindingbetween MZB1 and RACK1. An example of such an inhibitor is antibody 2F9.In another embodiment, the inhibitor interacts with the domain of MZB1which is involved in the regulation of cell proliferation and BCRsignaling and interferes with its function. In yet another embodiment,the inhibitor is targeted to the cytoplasm or the plasma membrane.

The inhibitors of the present invention need to be providedintracellularly in order to exert their inhibitory effects. Theinhibitors of the present invention may be modified and/or formulated bya person skilled in the art using known methods in order to achievetheir intracellular delivery.

Certain inhibitors, such as shRNA, scFv, dominant-negative MZB1 anddominant-negative interaction partner, may be provided in the form ofcoding nucleic acid molecules or vectors which allow for theirintracellular expression.

In a perferred embodiment, the inhibitors of the present invention aremodified and/or formulated for targetted delivery into B cells, inparticular, marginal zone (MZ) B cells, B-1 B cells, and/or follicular(FO) B cells.

In certain embodiments, the pharmaceutical composition is for use incombination with one or more existing treatments of autoimmune diseases.

Mammals include, but are not limited to, laboratory animals such asmice, rats, rabbits, cats, dogs, farm animals such as horses, sheep,cattle, cows, pigs, non-human primates, and humans. In a preferredembodiment, the mammal is human.

Use of an Activator or Enhancer of MZB1 Expression or Activity

The present invention provides an activator or enhancer of MZB1expression or activity for treating immunodeficiencies in a mammal, inparticular, immunodeficiencies that are caused by and/or associated withinsufficient antibody production.

The present invention also provides the use of an activator or enhancerof MZB1 expression or activity for the preparation of a pharmaceuticalcomposition for treating immunodeficiencies in a mammal, in particular,immunodeficiencies that are caused by and/or associated withinsufficient antibody production.

Immunodeficiencies include, but are not limited to, hereditaryimmunodeficiency, spontaneous immunodeficiency and drug-inducedimmunodeficiency (such as that induced by immunosuppressants used intransplantation and chemotherapeutic agents used for treating cancer).

The present invention further provides the use of an activator orenhancer of MZB1 expression or activity for enhancing antibodyproduction in a cell.

The present invention additionally provides an in vitro method forenhancing antibody production in a cell comprising contacting the cellwith an activator or enhancer of MZB1 expression or activity.

The cell may be a primary B cell, such as a FO B cell, a MZ B cell or aB1 B cell, a B cell line, or a B cell hybridoma.

Activators or enhancers of MZB1 expression include, but are not limitedto, nucleic acid molecules or vectors which are capable of driving theexpression of MZB1, a variant or a biologically active fragment thereof,and transcription factors which activate or enhance the transcriptionfrom an MZB1 coding sequence.

Activators or enhancers of MZB1 activity include, but are not limitedto, MZB1, a variant or a biologically active fragment thereof, agonisticantibodies, in particular, agonistic intrabodies, and small moleculeagonists.

In a preferred embodiment, the activator or enhancer activates orenhances only the antibody production-promoting activity of MZB1. In oneembodiment, the activator or enhancer interacts with the domain of MZB1which is involved in the regulation of antibody secretion and activatesor enhances with its function. In another embodiment, the inhibitor istargeted to the ER.

In a perferred embodiment, the activators or enhancers of the presentinvention are modified and/or formulated for targeted delivery into Bcells, in particular, marginal zone (MZ) B cells, B-1 B cells, and/orfollicular (FO) B cells.

The present invention additionally provides an activator or enhancer ofMZB1 expression or activity for treating immunodeficiencies in a mammal,in particular, immunodeficiencies that are caused by and/or associatedwith T cell deficiency due to reduced or absent TCR signalling and/orco-stimulation.

The present invention also provides the use of an activator or enhancerof MZB1 expression or activity for the preparation of a pharmaceuticalcomposition for treating immunodeficiencies in a mammal, in particular,immunodeficiencies that are caused by and/or associated with T celldeficiency due to reduced or absent TCR signalling and/orco-stimulation.

In a perferred embodiment, the activators or enhancers of the presentinvention are modified and/or formulated for targetted delivery into Tcells.

The activators or enhancers of the present invention need to be providedintracellularly in order to exert their activating or enhancer effects.The activators or enhancers of the present invention may be modifiedand/or formulated by a person skilled in the art using known methods inorder to achieve their intracellular delivery.

Certain activators or enhancers, such as MZB1, a variant or abiologically active fragment thereof, and agonist scFv, may be providedin the form of coding nucleic acid molecules or vectors which allow fortheir intracellular expression.

In certain embodiments, the pharmaceutical composition is for use incombination with one or more existing treatments for immunodeficiencies.

The present application provides MZB1 or a variant or a fragment thereofwhich comprises the amino acid sequence of SEQ ID NO:10 or an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:9 forinhibiting the growth of tumor cells and/or bacterial cells.

The present application provides MZB1 or a variant or a fragment thereofwhich comprises the amino acid sequence of SEQ ID NO:10 or an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:9 for treatinga tumor and/or a bacterial infection in a mammal.

Mammals include, but are not limited to, laboratory animals such asmice, rats, rabbits, cats, dogs, farm animals such as horses, sheep,cattle, cows, pigs, non-human primates, and humans. In a preferredembodiment, the mammal is human.

Method for Polypeptide Production

The present invention provides a process for producing a polypeptide ofthe present invention comprising culturing a host cell of the presentinvention under suitable conditions to express the polypeptide, andoptionally isolating the polypeptide from the culture.

The suitable conditions can be determined by a person skilled in theart, taking into consideration the host cell and expression vector used.

The polypeptide of the present invention may be enriched, partiallypurified or purified from the cultured host cells or the culture mediaby any suitable methods known in the art, such as those described in theCurrent Protocols in Protein Science (supra).

In some cases, the polypeptide of the present invention may not bebiologically active upon isolation. Various methods known in the art for“refolding” can be used to restore biological activity.

Method for Screening for Modulators of MZB1 Expression or Activity

The present invention provide a method for screening for moduclators,including inhitors, activator and enhancers, of MZB1 expression,comprising the steps of:

-   (a) contacting a cell expressing MZB1 or a variant thereof with    candidate compounds;-   (b) comparing the expression of MZB1 in the presence and absence of    the candidate compounds; and-   (c) identifying compounds which modulate (increase or decrease) MZB1    expression.    The present invention also provides a method for screening for    moduclators, including inhitors, activator and enhancers, of MZB1    activity comprising the steps of:-   (a) contacting MZB1, a variant or a biologically active fragment    thereof, or a cell expressing MZB1, a variant or a biologically    active fragment thereof with candidate compounds;-   (b) comparing the activity of MZB1 in the presence and absence of    the candidate compounds; and-   (c) identifying compounds which modulate (increase or decrease) MZB1    activity.

The present invnention is illustrated by the following examples.

EXAMPLES 1. Materials and Methods 1.1 Cell Lines

All cell lines were maintained at 37° C. in a 5% CO₂ gassed atmosphere.If not stated otherwise, cell lines were propagated according to theinformation supplied by ATCC (www.atcc.org). Cells growing in suspensionwere maintained at a concentration between 1×10⁵ and 1×10⁶ cells/ml.Adherent cells were grown to confluence. Every 2-3 days they were splitby removal of media and subsequent incubation with a 0.25% trypsin-EDTAsolution (Gibco) for 1 minute. Upon detachment, fresh media was addedand cells were diluted 1:6 into new culture dishes. Stocks in liquidnitrogen were prepared by freezing 5×10⁶ cells/ml in FCS containing 10%DMSO. Samples were frozen at −80° C. in an isopropanol-containingfreezing box (Nalgene) and transferred to liquid nitrogen after 24hours.

K46: This murine, mature B lymphoblastoid cell line is 20% semiadherentand produces surface IgG_(2a)/kappa (Kim et al., 1979). For maintenancecells were cultured in RPMI 1640 media (PAA) supplemented with 10% (v/v)heat-inactivated FCS, 1% (v/v) PSG and 56 μM β-ME.

MZB1 knockdown K46 cells (shRNA): Using RNA interference, MZB1 wasknocked down in K46 cells using an shRNA which can be processed into anactive siRNA which targets the sequence of GCGAAAGCAGAGGCTAAAT (#inv2)in the MZB1 coding region. Two independent clones were generated,#inv2-10 and #inv2-29. As control, K46 cells were stably transfectedwith the pSuper.neo+GFP vector without an MZB1-specific hairpin. Twoindependent clones were generated, #pSup-13 and #pSup-17. Cells werecultured in RPMI 1640 media (PAA) supplemented with 10% (v/v)heat-inactivated FCS, 1% (v/v) PSG (Penicillin-Streptomycin-Glutamine)and 56 μM β-mercaptoethanol (β-ME). Before freezing, theses cells werepassaged in selection media additionally containing 1 mg/ml active G418.

MZB1 knockdown K46 cells with restored MZB1 expression (shRNA+MZB1):MZB1 expression was reconstituted in shRNA cells by stable transfectionof pIRES-MZB1-mut-puro which drives the expression of a mutant versionof MZB1 which is resistant to downregulation by shRNA #inv2. K46shRNA+MZB1 cells were cultured in RPMI 1640 media (PAA) supplementedwith 10% (v/v) heat-inactivated FCS, 1% (v/v) PSG and 56 μM β-ME. Toselect for stable integration of the plasmid, cells were passaged inselection media additionally containing 1 mg/ml active G418 and 1 μg/mlPuromycin.

1.2 Primary Cell Culture—Splenocytes and Peritoneal Cells

All primary cells (splenocytes and peritoneal cells) were cultured at37° C. in a 5% CO₂ gassed atmosphere. Cells were maintained at aconcentration between 5×10⁵ and 1×10⁶ cells/ml in RPMI 1640 media (PAA)supplemented with 10% (v/v) heat-inactivated FCS, 1% (v/v) PSG, 1% (v/v)Non-essential-Amino-Acids, 1% Sodium Pyruvate, 10 mM HEPES and 56 μMβ-mercaptoethanol (splenocyte medium). Stocks in liquid nitrogen wereprepared by freezing 5×10⁶ cells/ml in FCS containing 10% DMSO. Sampleswere frozen in a freezing machine (Custom Bio Genic Systems) accordingto the manufacturer's recommendations and transferred to liquidnitrogen.

1.3 Retroviral Transduction of B1/MZ B Cells with MZB1 miRNA andSubsequent Analysis of Antibody Secretion

For introduction of MZB1 miRNA into primary suspension cells (e.g. B1 Bcells or MZ B cells) retroviral transduction using the packaging cellline GP+E 86 (Markovitz et al., 1988) was performed. This packaging cellline produces the retrovirus encoding for MZB1-specific hairpins whichtarget the same MZB1 sequence GCGAAAGCAGAGGCTAAAT as #inv2 and secretesthe assembled virus into the supernatant, ready to infect the targetcells. To generate the packaging lines GP+E86-miRNA-MZB1 and GP+E86-MCS(MCS stands for multiple cloning site and is an empty vector forcontrol), 293T cells were transiently transfected with p-miRNA-MZB1, aretroviral vector containing MZB1-specific hairpin on the pMSCV-IRES-GFPbackbone, or the empty vector p-miRNA-MCS. The IRES-GFP-cassette wassubstituted with a combined GFP-pre-miRNA-cassette derived from theinvitrogen BLOCK-iT™ Pol II miR RNAi expression vector system. Theexpression of the GFP-pre-miRNA-cassette is driven by the retroviral5′-LTR. This system permits visual or automated selection of cellsexpressing the pre-miRNA through co-cistronic expression of GFP. In bothcases, the retroviral helper plasmid pEQPAM3, encoding for the viralgag, pol and env genes was co-transfected. 12 hours post transfection,the supernatant of 293T cells was filtered (0.45 μm), polybrene added toa final concentration of 2 μg/ml and transferred onto semi-confluentGP+E86 cells. The supernatant was exchanged following this procedureevery 12 hours and repeated four times. Retrovirus producing cells wereenriched by cell sorting for GFP⁺ cells.

For retroviral transduction of B1 and MZ B cells, GP+E86-miRNA-MZB1 orGP+E86-MCS cells (5×10⁶ cells/10 plate) were plated on gelatinizedtissue culture plates and cultured for 16 hours. 5×10⁶ FACS-sorted B1 Bcells or MZ B cells were resuspended in splenocyte media containing 2μg/ml polybrene and co-cultured for 36 h on confluent GP+E86-miRNA-MZB1or GP+E86-MCS feeder-layers. Transduced B1 B cells or MZ B cells wereenriched by FACS-sorting for GFP⁺ cells. To control for downregulationof MZB1 protein levels, an immunoblot using anti-MZB1 specific antibodywas performed with cells extracts of infected B1 and MZ B cells andcompared to control cells infected with empty vector.

To investigate antibody secretion behavior in primary cells (B1 or MZ Bcells) with reduced MZB1 protein levels, ELISA assays as well as ELISPOTexperiments were performed. For ELISA, the infected and FACS sortedcells (GFP⁺ cells) were plated on 24 well plates and incubated for up tothree days in splenocyte medium in the presence or the absence of LPSstimulation. The concentration of secreted antibody was determined insupernatant isolated 6 h, 24 h, 48 h and 72 h after culture start usinga standard IgM-specific ELISA protocol. Moreover, modified ELISAprotocols specifically detecting polyreactive antibodies were carriedout, testing whether MZB1 reduction results in a shift of quality of thesecreted antibody. To address potential limitations in the ability of anELISA to detect the output of rare antibody secreting cells, ELISPOTassays which are theoretically able to detect every single cellsecreting IgM were performed. ELISPOT assays were carried out onMultiScreen-HA fluter plates (Millipore). GFP⁺ cells were incubated for6 h, 24 h, 48 h and 72 h at 37° C. on pre-coated 96-well filter platesin the presence or the absence of LPS stimulation and developed with APsubstrate, marking every antibody secreting cell with a blue spot on thefilter. Spots were counted using specific software in order to identifypossible differences in the number of antibody secreting cells betweenthe miRNA culture and the control culture of primary cells.

1.4 AnnexinV Staining

Annexin V staining for identification of early apoptotic cells wasperformed according to the protocol provided by the manufacturer (BDBiosciences). Briefly, cells were washed twice with cold PBS and thenresuspended in 1× Binding Buffer (supplied by manufacturer) at aconcentration of approximately 1×10⁶ cells/ml. 100 μl of the solution(˜1×10⁵ cells) were transferred to a 4 ml FACS tube. 5 μl Annexin V-PEand 5 μl 7AAD (vital dye) were added. The cells were gently mixed and anincubated for 15 min at room temperature in the dark. Afterwards 40001of 1× Binding Buffer were added to each tube and the samples wereanalyzed within 1 hour by flow cytometry.

1.5 Calcium Mobilization

Cells (5×10⁶) were incubated with 5 μg/ml of Indo-1 AM (MolecularProbes) and 0.5 μg/ml of nonionic, low-toxicity detergent Pluronic®F-127 (Molecular Probes) in RPMI medium (PAA) supplemented with 1% FCSat 37° C. After 45 min incubation, the cell pellets were resuspended inRPMI medium plus 1% FCS and kept on ice. Ca²⁺-response was induced byadding goat anti-kappa antibody (mouse-specific) (SouthernBiotechnology) at a final concentration of 5 μg/ml. TheCa²⁺-concentration of the used RPMI-media was either 2 mM or 0.5 mM.Increases in free intracellular calcium in gated B or T cell populationswere measured in real time on a FACSAria (BD Biosciences).

1.6 Proliferation Assay

Purified primary cells as well as cell culture suspension cells wereplated in triplicate in 96-well-flat-bottomed plates in concentrationsof 1×10⁵, 3×10⁴, 1×10⁴ and 3×10³ cells/well. Cells were either keptunstimulated or activated with the following mitogens: 5 μg/mlanti-kappa (Southern Biotechnology), 0.3 μg/ml anti-CD3ε (nano Tools),0.1-0.3 μg/ml anti-CD28 (eBioscience) or 2 ng/ml recombinant mouse IL-2(R&D Systems). Cells were pulsed with [³H]thymidine (1.5 μCi/well) for18 h. Proliferation as the mean [³H]thymidine incorporation oftriplicate cultures was assayed by scintillation counting.

1.7 Preparation of Whole Cell Protein Extracts

Cell pellets of stimulated or unstimulated cells were washed once withice cold 1×PBS and resuspended in an appropriate volume (50 μl-2 ml) ofRIPA buffer (50 mM Tris-Cl, pH8.0, 150 mM NaCl, 1.0% (v/v) NP-40 (IgepalCA-630), 0.5% deoxycholate (DOC), 0,1% SDS), Standard CoIP buffer (50 mMTris-Cl, pH 7.4, 15 mM EGTA, 100 mM NaCl, 0.1% (v/v) Triton X-100), orDigitonin-lysis buffer (50 mM Tris-Cl, pH7.5, 150 mM NaCl, 1% Digitonin,5 mM EDTA), freshly supplemented with 1×PMSF, 1× Sodium orthovanadate,1×PIM (protease inhibitor mix) and 1×NaF. For enhanced disruption ofcells and sheering of DNA, samples were sonified 2 times for 1 minute ina Branson sonifier 450 using a pre-chilled water bath, 100% duty and anoutput control of 6-7. In case of lysis with Digitonin-containingbuffer, sonification step was omitted. Complete cell lysis was checkedunder the microscope and the sample was centrifuged at maximum speed and4° C. for 15 minutes in an Eppendorf 5415R centrifuge to spin down celldebris. Protein content of the supernatant was determined by Bradfordassay as described, and the samples were either snap-frozen in liquidnitrogen and then stored at −80° C., or immediately used forimmunoprecipitation or immunoblot.

1.8 Membrane-Cytosol Fractionation

Membrane and cytosol fractions of K46 cells were prepared as previouslydescribed (Huber et al., 2000). Briefly, 1-5×10⁷ cells were pelleted andresuspended in 1 ml of ice cold hypotonic lysis buffer (20 mM Tris-C, pH7.4, 10 mM EDTA, 5 mM Na₃VO₄, 10% v/v protease inhibitor cocktail(Sigma)), incubated for 5 min on ice, and sonicated with 15×1^(−s)strokes on ice using an ultrasonic cell disruptor (Branson). Cell debriswas removed by centrifugation at 3000 rpm for 5 min at 4° C. Thesupernatant was centrifuged at 100,000×g for 60 min at 4° C. in anultracentrifuge (Optima® LE-80K Ultracentrifuge, Beckman), using aSW55Ti rotor. The supernatant corresponds to the cytosol fraction andwas stored on ice. The pellet was washed twice with hypotonic lysisbuffer containing 150 mM of NaCl. The pellet was then resuspended in200-500 μl of hypotonic lysis buffer containing 0.2% NP-40 and 0.5%sodium deoxycholate (NP-40-DOC-buffer) by repeated vortex mixing. Afterincubating for 60 min at 4° C. on a shaker (1000 rpm) (Thermomixercomfort, Eppendorf), the suspension was ultracentrifuged at 100,000×gfor 30 min, and the supernatant was collected as the membrane fraction.Protein contents of both cytosolic and membrane fractions weredetermined by Bradford assay as described, and the samples weresnap-frozen in liquid nitrogen and then stored at −80° C. After gentlythawing the samples on ice, they were used either forimmunoprecipitation or immunoblot.

1.9 Immunoprecipitation

Protein extracts were prepared as described. 500 μg-2 mg of proteinextract as determined by Bradford assay was used for a singleimmunopreciptation experiment. 1-5 μg of antibody was added and thetotal volume adjusted to 400 μl using Standard CoIP buffer (50 mMTris-Cl, pH 7.5, 15 mM EGTA, 150 mM NaCl, 0.1% (v/v) Triton X-100),Digitonin lysis buffer (50 mM Iris-Cl, pH7.5, 150 mM NaCl, 1% (w/v)Digitonin, 5 mM EDTA) or NP-40-DOC-buffer (20 mM Tris-C, pH 7.4, 10 mMEDTA, 0.2% NP-40, 0.5% sodium deoxycholate (DOC)), freshly supplementedwith 1×PMSF, 1× Sodium orthovanadate, 1×PIM (protease inhibitor mix) and1×NaF. The lysates were incubated on a rotary shaker at 4° C. for 5-16hours. 40 μl of Protein A, Protein G or Protein L beads that had beenblocked with 5% (w/v) BSA for 5-16 hours and equilibrated in theappropriate buffer were added and antibody capturing was performed for1-1.5 hours on a rotary shaker at 4° C. Beads were spun down at 800×gfor 3 minutes at 4° C. and washed 4 times with 1 ml of the buffer usedfor binding. Beads were resuspended in 40 μl 2×SDS loading dye andimmune complexes were eluted by boiling at 95° C. for 10 minutes.Samples were stored at −20° C. or immediately loaded onto an SDSpolyacrylamide gel.

When proteins tended to stick to the beads, a pre-clear step wasincluded before the addition of antibodies. For this, the proteinextract was supplemented with 40 μl of Protein A, Protein G or Protein Lbeads that had been equilibrated in the appropriate buffer and wasincubated on a rotary shaker at 4° C. for 2 hours. Afterwards, thesample was centrifuged at 800×g and 4° C. for 3 minutes. The supernatantwas transferred to a fresh 1.5 ml microcentrifuge tube, and used for theimmunoprecipitation procedure.

In order to reach higher precipitation efficiency, Protein A/G-purifiedanti-MZB1 antibodies 2F9, 5C11, 5H8 and 2H7 were directly coupled toCNBr-activated Sepharose-beads (GE Healthcare) and used for a “one-step”anti-MZB1 immunoprecipitation.

1.10 SDS PAGE and Immunoblot

Protein samples dissolved in 1×SDS loading dye were separated on SDSpolyacrylamide gels of the required resolution range. Gels were preparedaccording to standard procedures. Typically, MZB1 (≈21 kDa) was detectedon a 12% or 15% gel, whereas bigger proteins like PLCγ (≈150 kDa) orwhole cell extracts for α-phospho-tyrosine blots were separated on a 10%gel. Electrophoresis conditions were 200V and 400 mA for 45-60 minutesusing the appropriate buffer. Proteins were transferred onnitrocellulose membranes using semi-dry blot system (Bio-Rad). Transferconditions were 0.8 mA/cm2 and 300V for 1 hour in 1×Tris-Glycine buffer(2.5M Tris-Cl, 1.9M glycine, 1% (w/v) SDS) containing 20% methanol.Membranes were blocked for either 1 hour or o/n in 5% (w/v) non-fatdried milk powder dissolved in 1×PBS supplemented with 0.1% Tween®20(PBST). After a quick washing step using PBST, membranes were incubatedwith primary antibody diluted in PBST for 1-5 hours. The followingdilutions were used:

Antibody Dilution α-MZB1 (clone 2F9), rat monoclonal 1:100 α-PKC-β(clone 36), mouse monoclonal, #610127 1:200 α-RACK1 (clone 20), mousemonoclonal, IgM, #610177 1:200 α-Phospho-Btk (Tyr223), rabbit monoclonal 1:4000 α-Phospho-Btk (Ser180), mouse monoclonal  1:1000 (clone 3D3)α-Btk, rabbit monoclonal  1:2000 α-Phospho-PLCγ2 (Tyr1217), rabbitpolyclonal  1:1000 α-PLCγ2, rabbit polyclonal  1:1000 α-BAP32, mousemonoclonal 1:500 α-BIP, rabbit polyclonal 1:500 α-GAPDH (clone 6C5),mouse monoclonal  1:2000

Membranes were washed 3 times for 5 minutes with PBST and incubated for1 hour with secondary antibody conjugated to horseradish peroxidase.Secondary antibodies were diluted as followed:

Antibody Dilution goat α-mouse-IgG, peroxidase-conjugated 1:10000mouse-α-rat-IgG, peroxidase-conjugated 1:3333  goat-α-mouse-IgM,peroxidase-conjugated 1:10000 goat-α-rabbit-IgG, peroxidase-conjugated1:10000

Membranes were washed 3 times for 5 minutes with PBST before beingincubated for 2 min with ECL™ Western Blotting Detection Reagents(Amersham/GE Healthcare). For visualization of proteins, membranes wereexposed to autoradiographic films (Bechtold) for different time periods.

1.11 Immunofluorescence

In order to determine the exact location of overexpressed and endogenousproteins within a cell, immunostaining on fixed cells was performed.Images of the fixed slides were taken on a LSM 510 META confocalmicroscope (Zeiss) using a 63×/oil objective.

1.11.1 Indirect Immunofluorescence of Adherent Cells

Cover slips were placed in 6-well tissue culture plates and coated withan excess of Poly-L-lysine solution (diluted 1 to 10 in ddH₂O) for 10minutes at room temperature. The Poly-L-lysine solution was aspired andafter drying the coverslips at 70° C. for 1 hour, 2×10⁵ cells/well ofNIH 3T3-FLAG-MZB1 cells (NIH3T3 cells stably expressing N-terminallyFLAG-tagged MZB1 under the control of the CMV promoter) were seeded onthe coated coverslips. Cells were cultured as previously described for16-24 hours.

Cells were washed once with PBS and fixed for 10 minutes with 2 ml of 4%(w/v) paraformaldehyde solution. After fixation, cells were washed 3times for 5 minutes with 1×PBS containing 0.1% Triton X-100. Blockingwas performed in 1×PBS containing 3% (v/v) goat serum for 30 min.Subsequently, primary antibody diluted in 100 μl 1×PBS, 0.1% TritonX-100 and 3% goat serum was added to the cells, and the cover slips werecovered with a small parafilm strip to prevent drying. After 1 hourincubation, the cover slips were washed 3 times for 5 minutes with 1×PBScontaining 0.1% Triton X-100. Incubation with secondary antibody diluted1 to 100 in 1×PBS, 0.1% Triton X-100 and 3% goat serum was performed for1 hour in the dark. After 3 washing steps of 5 minutes each, cell nucleiwere stained with 0.5 ml of 5 μg/ml DAPI diluted 1 in 1000 in 1×PBS,0.1% Triton X-100 for 5 minutes. Cells were mounted on slides usingProLong® Gold antifade reagent (Invitrogen). After incubation for 24hours at room temperature in the dark, slides were sealed with nailpolish. The following dilution of primary and secondary antibody wasused:

Antibody Dilution α-MZB1 (clone 2F9), rat monoclonal undiluted α-BAP31,rabbit monoclonal 1:100 Alexa Fluor ® 647 chicken α-rat IgG 1:200 AlexaFluor ® 488 goat α-rabbit IgG 1:200

1.11.2 Indirect Immunofluorescence of Suspension Cells

Covers lips were placed in 6-well tissue culture plates and coated withundiluted Poly-L-Lysine for 10 minutes at room temperature. Shortlybefore use the plates were washed with 1×PBS for 5 to 20 minutes andthen rinsed with RPMI 1640 (PAA) complete media. Suspension cells (e.g.MZ B cells) were centrifuged and resuspended in an appropriate amount ofmedia. 3×10⁵ cells in 100 μl volume were dropped on each cover slip andincubated for 3-5 minutes at room temperature to let the cells settledown.

Further steps were performed accordingly to the procedure for adherentcells. The following dilution of primary and secondary antibody wasused:

Antibody Dilution α-MZB1 (clone 2F9), rat monoclonal undiluted α-RACK1(clone 20), mouse monoclonal, IgM 1:200 Alexa Fluor ® 647 chicken α-ratIgG 1:200 Alexa Fluor ® 488 goat α-mouse IgM 1:200

1.12 MZB1 Knock-Out Mouse

A pGKneo-pA cassette was inserted in frame with the ATG codon of themzb1 gene, which renders mzb1 inactive (FIG. 37). Homologousrecombination in ES cells was identified by Southern blot analysis. Therelevant restriction sites used for Southern blot analysis and thelocation of the probe are indicated (FIG. 37).

1.13 Retroviral Transduction of Primary Follicular B Cells with MZB1Expression Vector

For stable introduction of MZB1 cDNA into primary suspension cells (e.g.follicular B cells) retroviral transduction using the packaging cellline GP+E 86 (Markowitz et al., 1988) was performed.

To generate the packaging lines GP+E86-MZB1 and GP+E86-MCS, 293T cellswere transiently transfected with pEG2-MZB1 (retrovirus containing MZB1cDNA) or the empty vector pEG2-MCS. In both cases, the retroviral helperplasmid pEQPAM3 encoding the viral gag, pol and env genes wasco-transfected. 12 h post transfection, the supernatant of 293T cellswas filtered (0.45 μm), polybrene added to a final concentration of 2μg/ml and transferred onto semi-confluent GP+E86 cells. The supernatantwas exchanged following this procedure every 12 h and repeated fourtimes. Retrovirus producing cells were enriched by cell sorting for GFP⁺cells.

For retroviral transduction of follicular (FO) B cells, GP+E86-MZB1 orGP+E86-MCS cells (5×10⁶ cells/10 plate) were plated on gelatinizedtissue culture plates and cultured for 16 h. 5×10⁶ FACS-sortedfollicular B cells were resuspended in splenocyte media containing 2μg/ml polybrene and co-cultured for 36 h on confluent GP+E86-MZB1 orGP+E86-MCS feeder-layers. Transduced FO B cells were enriched byFACS-sorting.

1.14 Transient Transfection of Adherent Cells Using Calcium Phosphate

For a single transfection, 3.2×10⁵ GP+E 86 were seeded onto a 6 cmtissue culture plate 14 hours before transfection. 250 μl of freshlyprepared 250 mM CaCl₂ were mixed with 15 μg of DNA (vector DNA+salmonsperm DNA to reach at least 15 μg). The solution was supplemented with250 μl HBS, pH7.05 (280 mM NaCl, 10 mM KCl, 1.5 mM Na₂HPO₄, pH7.5, 12 mMglucose, 50 mM HEPES, pH7.6) under stirring conditions. The mixture wasincubated for 25 minutes at RT before being added to the cells whosemedium had been replaced with 5 ml of fresh complete medium in themeantime. The transfection mixture was incubated for 4-6 hours. Toremove calcium phosphate precipitates, cells were washed three timeswith 1×PBS and upon addition of fresh medium, incubated for 36-48 hours.

1.15 Primary Cell Culture—Splenocytes and Peritoneal Cells

All primary B cells (splenocytes and peritoneal cells) were cultured at37° C. in a 5% CO₂ gassed atmosphere. Cells were maintained at aconcentration between 5×10⁵ and 1×10⁶ cells/ml in RPMI 1640 media (PAA)supplemented with 10% (v/v) heat-inactivated FCS, 1% (v/v) PSG, 1% (v/v)Non-essential-Amino-Acids, 1% Sodium Pyruvate, 10 mM HEPES and 56 μMβ-mercaptoethanol (splenocyte medium). Stocks in liquid nitrogen wereprepared by freezing 5×10⁶ cells/ml in FCS containing 10% DMSO. Sampleswere frozen in a freezing machine (Custom Bio Genic Systems) accordingto the manufacturer's recommendations and transferred to liquidnitrogen.

1.16 Staining of Primary B Cells for FACS Sorting

Peritoneal cells and erythrocyte-depleted splenocytes from 1.5-4 monthold mice (C57BL/6J; Jackson Laboratories) were isolated. The cells werewashed twice in FACS buffer (2% FCS in 1×PBS) and the pellet wasresuspended in an appropriate volume of FACS buffer.

Cells from spleen and peritoneum were stained with antibody compositionswhich allow for the identification and isolation of B-1, follicular B(FO) and marginal zone (MZ) B cell subpopulations. For triple staining,optimized dilutions of fluorescein isothiocyanate (FITC)-, phycoerythrin(PE)-, and allophycocyanin (APC)-conjugated antibodies were used. Toassure highly specific staining, all specific antibodies used wereanti-mouse antibodies.

For unstained and single-stained reference samples, approximately0.5×10⁶ cells were taken out to each well of a round-bottomed 96-wellplate. The remaining cells were transferred to FACS tubes. Aftercentrifugation the pellets were resuspended and incubated for 10 min onice in a 1:200 dilution of F_(C)R (a-CD16/CD32) block in FACS buffer totarget the F_(C) receptors on myeloid and B lymphoid cells, which innon-blocked state unspecifically bind the antibodies employed for FACSanalysis. The samples were centrifuged, the blocking solution wasflicked out, and the cells were incubated in primary antibody in FACSbuffer for 20-40 min on ice. The cells were washed three times in FACSbuffer and, if necessary, with secondary antibody in FACS buffer foranother 20-40 min on ice. After three washing steps cells weretransferred into FACS tubes through a filter. After sorting, cells werewashed once, and either cultured as described or the cell pellet wasfrozen at −80° C. If not stated differently, all FACS antibodies usedwere purchased from BD Pharmingen.

1.17 ELISA and ELISPOT

To investigate antibody secretion behavior in primary FO B cells withincreased MZB1 protein levels, ELISA assays as well as ELISPOTexperiments were performed. For ELISA, the infected and FACS sortedcells (GFP⁺ cells) were plated on 24 well plates and incubated for up tothree days in splenocyte medium in the presence or the absence of LPSstimulation (1 μg/ml). The concentration of secreted antibody wasdetermined in supernatant isolated 6 h, 24 h, 48 h and 72 h afterculture start using a standard IgM-specific ELISA protocol. Moreover,modified ELISA protocols specifically detecting polyreactive antibodieswere carried out, testing whether an increase in MZB1 results in a shiftof quality of the secreted antibody. To address potential limitations inthe ability of an ELISA to detect the output of rare antibody secretingcells, ELISPOT assays which are able to detect every single cellsecreting IgM were performed. ELISPOT assays were carried out onMultiScreen-HA filter plates (Millipore). GFP⁺ cells were incubated for6 h, 24 h, 48 h and 72 h at 37° C. on pre-coated 96-well filter platesin the presence or the absence of LPS stimulation (1 μg/ml) anddeveloped with AP substrate, marking every antibody secreting cell witha blue spot on the filter. Spots were counted using specific software inorder to identify possible differences in the number of antibodysecreting cells between wt FO B cells and FO B cells overexpressingMZB1.

1.18 Generation of T Cell-Specific MZB1 Transgenic Mice: Lck-MZB1-M3,Lck-MZB1-F38 and Lck-MZB1-F47

The MZB1 transgenic construct was generated by inserting PCR-amplifiedmouse MZB1 genomic DNA into the BamHI site of the vector plck-hGH(Garvin et al., 1990), which contains the T-cell-specific Ick proximalpromoter and the human growth hormone (hGH) gene with introns and apolyadenylation signal. Plasmid DNA was linearized using a NotIrestriction digest, followed by agarose gel extraction and microinjectedinto FVB-derived zygotes. Transgenic founders were identified bySouthern blot analysis of the tail genomic DNA according to standardprotocols. Three independent transgenic lines were established andbackcrossed with C57BL/6J wt-mice. Transgenic offspring were determinedby PCR of the tail genomic DNA with the transgene-specific primers. Forvarious experiments, transgenic offspring and their wt littermates ofthe three independent lines were analyzed.

2. Results 2.1 Analysis of the expression pattern of MZB1 and itssubcellular localization 2.1.1 MZB1 is expressed specifically in B cells

MZB1 was initially identified as a lymphoid-specific cDNA clone from amurine 70Z/3 pre-B cell bacteriophage library by differential screening.In vivo, MZB1 is present in secondary lymphoid tissues and theexpression of MZB1 was found to be highest in the subpopulations of Bcells, whilst only minor MZB1 expression was detected in FO B cells(FIGS. 1A+B). MZB1 mRNA was detected in cultured cell lines of allstages of B cell development including fetal liver- or adult bonemarrow-derived pre-B, B and plasmacytoma cell lines, but absent fromcell lines of the T, myeloid, fibroblastic or erythroid lineages. Atissue Northern blot analysis on poly-A⁺ RNA from different mousetissues revealed that MZB1 transcript is confined to tissues containingB cells, such as the spleen and lymph nodes.

To facilitate the analysis of MZB1 protein expression, four differentrat monoclonal α-mouse MZB1 antibodies were generated.Immunohistochemistry and immunoblot analysis using these antibodiesrevealed abundant MZB1 protein in the spleen and peritoneum. TheMZB1-expressing cells in these tissues are B220^(dull) and IgM^(high),indicating that they belong to a subset of activated B cells.

2.1.2 MZB1 Protein is Highly Expressed in Splenic Marginal Zone B Cells(MZ) and Peritoneum Derived B1 B Cells

Different B cell populations originating from the bone marrow, spleenand peritoneal cavity (PC) were FACS-sorted and an MZB1 immunoblotanalysis was performed on equal amounts of protein (FIG. 1B).

MZB1 expression is high in splenic MZ B cells and peritoneal cavityderived B1 B cells, but low to absent in the splenic follicular B cells(FO). The immature B cell subsets represented by bone marrow derivedfraction B and C as well as splenic transitional 1 and 2 B cells (T1/2)display a moderate expression level of MZB1 protein (FIG. 1B).

2.1.3 Intracellular Localization of MZB1 Protein

Computational analyses of the MZB1 amino acid sequence suggest that MZB1contains a signal peptide targeting the protein to certain intracellularcompartments and possibly to the secretory pathway. Furthermore, theMZB1 ORF contains a weak putative ER retention signal, raising thequestion whether MZB1 protein is retained in the endoplasmic reticulum(ER). To investigate the intracellular localization of MZB1, indirectimmunofluorescence experiments, visualized with a confocal microscope,were performed using FACS-sorted MZ B cells (FIG. 2).

In primary MZ B cells, MZB1 protein was found to localize to thecytoplasm throughout the cytosol, around the nuclear envelope and thecytoplasmic membrane (FIG. 2: upper left and lower right panel). In someregions of the cell, MZB1 is restricted to a punctuate, cytoplasmicpattern, indicating that a fraction of the cellular MZB1 pool isretained in specific cytosolic compartments, likely the endoplasmicreticulum. MZB1 appeared to be entirely excluded from the nucleus (FIG.2).

Since MZ B cells are very small in size, consisting of a predominantnucleus surrounded by a thin layer of cytoplasm, these cells are notoptimal to study the localization of a cytoplasmic protein like MZB1using confocal microscopy. To overcome these obstacles, indirectimmunofluorescence experiments were performed using a NIH 3T3 murinefibroblast cell line stably expressing FLAG-MZB1. These MZB1-expressingfibroblastic cells are larger in size compared to MZ B cells and as theyare widely spread on the surface, cellular compartments can bevisualized more accurately using confocal microscopy (FIG. 3).

MZB1 was also found to be expressed in a punctuate, cytoplasmic patternin NIH 3T3 FLAG-MZB1 cells (FIG. 3A, B), suggesting ER distribution. Thenuclear membrane was stained as well.

An antibody specifically for BCR-associated protein of 31 kDa (BAP31),which is an ubiquitously expressed ER-localized polytopic membraneprotein harboring three putative transmembrane (TM) regions, wasutilized for visualizing the ER of NIH 3T3 FLAG-MZB1 cells (FIG. 3A, C).The comparison of the MZB1 and BAP31 staining patterns shown in FIG. 3revealed a high degree of colocalization of the two proteins in the ERcompartment surrounding the nucleus. In areas more distant from thenucleus, the staining patterns of MZB1 and BAP31 did not show asignificant overlap (FIG. 3A, B and C). In these parts of the cell, MZB1appeared to be evenly dispersed within the cytosol. These resultssuggest the possible existence of at least two pools of MZB1 proteinwithin the cell, one of which is localized to the ER whereas the otherone is distributed in the cytosol. Furthermore, as in MZ B cells (FIG.2), MZB1 staining was also found at the cytoplasmic membrane in NIH 3T3FLAG-MZB1 cells (FIG. 3A, B), suggesting a possible association of MZB1protein with the cell membrane.

Further experiments, using confocal microscopy in combination withnocodazole treatment of the cells (FIG. 4) as well as biochemicaltechniques, namely proteinase K digestion of subcellular fractions (FIG.5), revealed the signal peptide and ER-retention motif containingprotein MZB1 to be localized in the lumen of the endoplasmaticreticulum, most probably associated with the ER membrane.

2.1.4 MZB1 Protein is Associated with the Cell Membrane

To investigate the subcellular localization of MZB1 in more detail,cytosolic and membrane fractions of K46 cells were prepared by hypotoniccell lysis and subsequent ultracentrifugation. After resuspending thewashed membranes in Nonidet-P40 (NP40) and sodium deoxycholate(DOC)-containing lysis buffer, an equal amount of protein from eachfraction was used in an MZB1-specific immunoblot (FIG. 6). To controlfor a possible cross contamination between the membrane fraction and thecytosolic fraction, a GAPDH-(cytosolic protein) and a BAP32-specificimmunoblot were also performed. BCR-associated protein of 32 kDa (BAP32)is a highly conserved, ubiquitously expressed protein that mainlylocalizes to the mitochondrial membrane.

About 90% of total MZB1 was found to be in the cytosolic fraction innon-stimulated K46 cells. The remaining 10% of MZB1 was found to beassociated with cellular membranes. The control immunoblots withantibodies specific for GAPDH and BAP32 revealed no cross contaminationbetween of the two fractions (FIG. 6).

This result is in agreement with those obtained from MZB1immunofluorescence experiments (FIGS. 2 & 3) that a small pool of MZB1protein is associated with cellular membranes, likely the plasmamembrane, whereas the majority of MZB1 is localized in the cytosolic ofnon-stimulated cells (FIG. 6).

To further characterize the subcellular localization of MZB1,BCR-stimulated K46 mature B cells were fractionated into cytosolic andmembrane fractions (FIG. 7). The cells were serum-starved for 1 h andsubsequently stimulated with 5 μg/ml α-kappa antibody (SouthernBiotechnology) for 0 min, 1 min, 5 min and 20 min.

Consistent with the results presented in FIG. 6, in non-stimulated K46 Bcells, the majority of MZB1 (70%) is present in the cytosolic fraction.Upon stimulation of the BCR with α-kappa, a significant amount of MZB1is recruited to the membrane fraction, changing the ratio to 35%cytosolic and 65% membrane associated MZB1 (FIG. 7). An extension of thestimulus to 5 min or 20 min leads to a decrease in the amount of MZB1associated with the membrane component, which is additionally revealedby a densitometric analysis of the immunoblot signals (FIG. 8). Sincethe amount of cytosolic MZB1 is kept nearly constant during the timecourse from 0 min to 5 min, it is likely that the weak MZB1 signal inthe cytosolic fraction after 20 min of stimulation is caused by a poortransfer efficiency during the immunoblot procedure (FIG. 7). This wouldexplain the recurrence of a switch to 33% cytosolic and 66%membrane-bound MZB1.

The subcellular fractionation of K46 cells into cytosolic and membranecomponents indicates that in non-stimulated cells, between 10% to 30% ofthe cellular MZB1 pool is associated with the membrane fraction. UponBCR-stimulation, MZB1 protein is dynamically recruited to the membranefollowed by a decrease in MZB1 membrane association after extendedstimulation. These results are confirmed by immunofluorescenceexperiments, showing a weak staining of the cytoplasmic membrane innon-stimulated cells (FIG. 2, 3) and an evident recruitment of MZB1 tothe cell membrane after BCR-stimulation.

2.2 Search for Interaction Partners 2.2.1 Yeast Two Hybrid Screen withMZB1

Apart from the biochemical complex purification, a high throughputscreening in form of a yeast two hybrid screen to identify novelinteraction partners of MZB1 was performed. Briefly, the bait protein,in this case MZB1, is fused to the Gal4 DNA-binding domain and binds toa promoter which has to be active in order to permit growth underselection conditions. A human B cell library as well as a human T celllibrary was cloned into an expression vector containing the Gal4activation domain serving as a prey (Durfee et al., 1993). If bait and apotential interaction partner, the prey, come together, the Gal4activation domain turns on transcription of a selection marker. Thevector DNA of single clones can be isolated and sequenced.

For the screen, the murine cDNA of MZB1 was cloned into the yeast pGBT9expression vector and obtained a number of clones which were isolatedand sequenced (Tables 1 & 2).

TABLE 1 Clones obtained from T cell library: Definition Number of clonesRACK1 (Receptor for activated C kinase; 17 Lung cancer oncogene 7)Serpinpeptidase-inhibitor (Ovalbumine) 1 Dehydrogenase/reductase 1(SDR-family) 1 Methylenetetrahydrofolate dehydrogenase 1 (NADP +dependent) Succinate dehydrogenase complex 1

TABLE 2 Clones obtained from B cell library: Definition Number of clonesRACK1 (Receptor for activated C kinase; 10 Lung cancer oncogene 7)Immunoglobulin μ heavy chain 3 RUNX3 (Runt-related transcription factor3; 1 PEBP2aC1; AML2) Ku70-binding protein 3 1Nascent-polypeptide-associated complex alpha 1 polypeptide (α-NAC)Component of oligomeric golgi complex 4 1

Among the potential interaction partners obtained in two independentyeast two hybrid screens using a human T cell library or a human B celllibrary, one protein, namely “receptor for activated C kinase” (RACK1)turned out to be predominant. RACK1, a scaffolding protein consisting of7 WD40 repeats is an especially interesting protein by virtue of itsability to coordinate the interaction of key signaling molecules andhence playing a central role in the organization of critical biologicalprocesses like the regulation of proliferation, cell cycle and celladherence (McCahill et al., 2002). Interestingly, the two independentyeast two-hybrid screens did not yield BiP as a potential interactionpartner of MZB1 which could be explained by the fact that GRP78 was notpresent or underrepresented in the used libraries. Due to their smallnumbers of clones detected in the screen, the remaining hits wereneglected.

2.2.2 MZB1 Interacts with RACK1 and PKCβ in the Membrane Fraction of K46Cell Extracts

In order to confirm the results of the yeast two-hybrid screen,co-immunoprecipitation experiments were carried out. In a first attemptto verify the results obtained from the yeast two hybrid screen,standard CoIP conditions (Standard CoIP buffer freshly supplemented with1×PMSF, 1× Sodium orthovanadate, 1×PIM and 1×NaF) with whole cellextracts of BCR stimulated and untreated K46 B cells were used todemonstrate a potential interaction between MZB1 and RACK1 following thestandard co-immunoprecipitation protocol. Immunoprecipitation of bothendogenous proteins with their specific antibodies revealed direct IPsignals but co-immunoprecipitation was, however, hardly detected forboth cases (data not shown). Furhtermore, it was analyzed whether theinteraction of MZB1 and RACK1 was restricted to the membranecompartment. It was thought that the separation of whole cell extractsinto a membrane and a cytosolic compartment would enrich the potentialprotein complex in the subcellular fraction where it is located and sofacilitate detection. Membrane and cytosolic extracts of K46 B cellswere prepared as described before. CoIP was carried out using, persingle IP, 1 mg of total protein and either 3 μg of α-RACK1 antibody (BDBiosciences) or 40 μl of a 50% mixture of CNBr-activated Sepharose-beads(GE Healthcare) directly coupled to α-MZB1 antibody (clone 5C11) orα-BCL9 control antibody.

As can be seen in FIG. 9, the direct IP signal for RACK1 and MZB1 couldbe detected in the cytosolic as well as the membrane fraction of K46 Bcells. The co-immunoprecipitation (co-IP) signal for both RACK1 and MZB1was absent from the cytosolic but present in the membrane fraction (FIG.9). Taken together, the co-immunoprecipitation results described hereconfirm the interaction between MZB1 and RACK1 suggested from the yeasttwo-hybrid screens performed with a human B cell and T cell library(Tables 1 & 2). Furthermore, the separation of K46 whole cell extractinto a cytosolic and a membrane fraction reveals that the MZB1-RACK1association is restricted to the membrane compartments of the cell (FIG.9). These results are in accordance with the finding that innon-stimulated K46 cells, approximately 10% of MZB1 protein isassociated with cellular membranes (FIGS. 6 & 7). The membrane pool ofMZB1 seems to be associated with the membrane pool of RACK1. Moreover,to confirm the association of MZB1 and RACK1 in a further set ofexperiments, FRET (Fluorescence Resonance Energy Transfer) microscopyanalysis was performed, suggesting a direct interaction between MZB1 andRACK1 (data not shown).

Since RACK1 was shown to interact, amongst various other proteins, withprotein kinase C β (PKCβ) (Ron et al., 1994), co-IP experimentsprecipitating PKCβ from cytosolic as well as membrane extract derivedfrom non-stimulated K46 B cells were performed. The procedure forpreparation of cytosolic and membrane fraction as well as the protocolfor the immunoprecipitation were the same as the α-MZB1 and α-RACK1 IPsdescribed above. Per single IP, 1 mg of total protein and 3 μg of α-PKCβantibody (BD Biosciences) were used.

As revealed in FIG. 10, RACK1 and PKCβ interact with each other in thecytosolic as well as the membrane fraction of K46 B cells, confirmingthe already published association of the two proteins (Ron et al.,1994). In accordance with the results of the IPs performed with α-MZB1and α-RACK1 antibodies, a co-IP signal for MZB1 was detected in themembrane but was absent from the cytosolic fraction.

In summary, these results indicate an interaction of MZB1 with RACK1 andMZB1 with PKCβ, in the membrane compartment of non-stimulated K46 Bcells (FIGS. 9 & 10), suggesting the formation of a ternary complexconsisting of MZB1, RACK1 and PKCβ. The association between MZB1 and BiPwas shown to be destabilized upon BCR stimulation. It remains to betested whether the stability of the interaction between MZB1 and RACK1or between MZB1 and PKCβ is affected by BCR stimulation. The fact thatthe interaction of MZB1, RACK1 and PKCβ is restricted to the membranefraction is consistent with the observation that the complexed form ofMZB1, which elutes at 220 kDa in gel filtration experiments, is foundspecifically in the membrane compartment but not the cytosoliccompartment of a whole cell extract (data not shown).

2.2.3 ERp57, GRP94, ERp44 and BiP are Potential Interaction Partners ofMZB1

In a further search for potential interaction partners of MZB1, K46cells were either kept untreated or crosslinked with 1% formaldehydeprior to cell lysis. Using MZB1-specific antibodies, MZB1 protein wasimmunoprecipitated from both crosslinked as well as untreated cellextracts, and a 4%-12% gradient SDS-PAGE followed by a silver stain wasperformed. Bands, specifically appearing in the lane corresponding tothe crosslinked cell extract, were excised and analyzed bymass-spectrometry (FIG. 11). Besides BiP, which was shown in previousexperiments to interact with MZB1, the potential novel interactionpartners, namely GRP94 (Endoplasmin), ERp57 and ERp44, being identifiedin the mass-spectrometry analysis, were confirmed to interact with MZB1by co-immunoprecipitation experiments, using antibodies generatedagainst the endogenous proteins (FIGS. 12 & 13).

ERp44: Interestingly, ERp44, a thioredoxin (TRX) family protein has notonly been implicated in oxidative protein folding, but has as well beenshown to specifically bind to the ER-luminal L3V domain of the inositol1,4,5-trisphosphate receptor 1 (IP₃R1), hence inhibiting theCa²⁺-channel activity of the IP₃R1 in a pH-, redox state-, and[Ca²⁺]_(ER)-dependent manner. The interaction of MZB1 with ERp44 raisesthe possibility that MZB1 might affect ER calcium stores (FIGS. 32, 35 &36A) through the ER-membrane localized Ca²⁺-channel IP₃R1.

ERp57: Supporting the notion of a calcium sensitive high molecularweight MZB1 complex, the interaction of ERp57 and MZB1 was shown to becalcium dependent (FIGS. 14 & 15A). Since ERp57, which belongs to theclass of protein disulfide isomerases (PDI), was shown to regulate SERCApump activity in a calcium dependent manner, it could be speculatedwhether MZB1 might be involved in the regulation of SERCA pump activityvia ERp57, explaining the observed effects of MZB1 on ER calcium stores.Furthermore, size exclusion chromatography experiments revealed thatboth MZB1 and ERp57 co-migrate at an approximate molecular weight of 220kDa in the absence of calcium, and both shift to lower molecular weightfractions if calcium was added to a final concentration of 2.5 mM to K46membrane extracts (FIG. 16).

GRP94: On the contrary, for GRP94, belonging together with BiP to amulti-chaperone complex, responsible for the correct folding of cellsurface proteins like various integrins as well as the Toll likereceptor 4 (TLR4), its MZB1 interaction was shown to be calciumindependent (FIGS. 14 & 15B). This fact raises the possibility of analternative, ER-localized protein complex comprising amongst others ofGRP94, BiP and MZB1. Interestingly, the cell surface expression of α4and β1 integrins was reduced in K46 siRNA cells, whereas β2 surfaceexpression was almost unchanged if compared to K46 wt and K46siRNA::MZB1 cells (FIG. 17). Thus, MZB1 affects cell-surface expressionof integrins on K46-siRNA cells. Considering the fact that the inducibleknock out of GRP94 in B cells shows specific defects in the folding ofintegrins and the TLR4, MZB1, belonging to the same multi-chaperonecomplex like GRP94, might be a potential regulator of integrin folding.This hypothesis is further supported by some modest changes in TLR4 andintegrin surface expression on MZB1-transduced FO B cells, compared toMock-transduced control FO B cells (FIG. 18). Thus, MZB1 affectscell-surface expression of integrins and TLR4 on MZB1-transduced FO Bcells.

2.2.4 The 220 kDa MZB1 Protein Complex is Calcium Sensitive

Cytosolic and membrane extracts of K46 mature cells were prepared in thepresence and in the absence of Ca²⁺. The above-described (section 2.2.2)high molecular weight MZB1 protein complex (220 kDa), which isspecifically found in the membrane fraction of K46 B cells, wasseparated by gel filtration, followed by an immunoblot analysis (FIGS.19 & 20). As shown in FIG. 20, the MZB1 protein complex was disrupted if2.5 mM CaCl₂ was added prior to cell lysis and membrane fractionpreparation. These results show that the high molecular weight MZB1protein complex (220 kDa) is calcium sensitive.

Taken together, the subcellualr localization of MZB1 in the ER, and thepresence of a calcium-sensitive high molecular weight complex, indicatethat MZB1 might be involved in the regulation of ER calcium stores,which are as well critical for the modulation of, e.g., BCR signallingand cell activation.

2.3 Functional Analysis of MZB1: Loss of Function 2.3.1 Generation ofMZB1 Knock-Down B Cells as a Tool to Study MZB1 Functions In Vitro

ShRNA was used to knock down MZB1 expression in K46 mature B cells inorder to investigate the effects of reduced MZB1 protein levels on cellproliferation and BCR signaling capacity. Oligonucleotides were designedusing the freely accessible “BD-RNAi design” (Beckton Dickinson) and“Block-iT RNAi” (Invitrogen) programs. The two most highly ratedoligonucleotides from each prediction program, #inv2:GCGAAAGCAGAGGCTAAAT, #inv3: GCAGTCCTATGGAGTTCAT, #BD1:CCAGATCTATGAAGCCTAC, and #BD4: CTGCCACTGTTGCTACTGT (all sequences listedare target sequences), were ordered and cloned into the pSuper RNAisystem (Oligo Engine) which allows for the transcription of smallhairpin RNAs (shRNA). All four oligonucleotides target the coding regionof MZB1 and were tested in transient transfection assays in K46 cells.ShRNA #inv2 knocked down MZB1 mRNA levels to 16.5% of the wild-type (wt)expression level, whereas the other shRNAs were less effective (#inv3:28.7%; #BD1: 36.1%; #BD4: 30%). The shRNA constructs additionallycontain the GFP coding sequence, the expression of which allows for cellsorting, as well as a neomycin resistance cassette, which enables theselection of cells that have stably integrated the exogenous construct.The construct pSuper.neo+GFP+inv2 containing the shRNA #inv2 wastransfected into K46 mature B cells and single cell clones weregenerated using FACS sorting followed by selection with neomycin. MZB1protein levels of individual clones were determined using immunoblotanalysis and four clones with significantly reduced MZB1 protein levelswere identified, one of which is displayed representatively in FIG. 21(lane 2). The differences in the extend of MZB1 knock-down observedbetween different clones were likely due to the integration sites of theconstructs.

Two stable K46 clones transfected with pSuper.neo+GFP+inv2, #inv2-29 and#inv2-10, which had the strongest downregulation of MZB1-protein levels,were used to investigate the phenotype of MZB1 loss-of-function.

In order to restore MZB1 expression in shRNA clones #inv2-29 and#inv2-10, cells were transfected with the “rescue” plasmidpIRES-MZB1-mut-puro which drives the expression of a mutated version ofMZB1 which is not affected by the MZB1-specific shRNA stably expressedby pSuper.neo+GFP+inv2. The bulk transfected population was selectedsimultaneously with puromycin and neomycin according to standardconditions. In all experiments performed, K46 shRNA cells (shRNA) werecompared with the corresponding “rescued” clone (shRNA+MZB1) and K46cells stably transfected with the empty vector pSuper.neo+GFP referredto as “wt” (wild-type). All experiments were carried out with clone#inv2-29, #inv2-10 and their corresponding “rescued” cells with restoredMZB1 expression. Since similar results were obtained for #inv2-29 and#inv2-10, results obtained with clone #inv2-29 and its correlated“rescued” clone are displayed unless otherwise indicated.

The immunoblot analysis of MZB1 protein levels reveals a reduction byapproximately 95% in shRNA cells compared to the wt (FIG. 21, lanes1+2), indicating a successful knock down of MZB1. In shRNA+MZB1 cells,the level of MZB1 protein was comparable to the wt level, suggesting afull restoration of MZB1 protein levels in the “rescued” cells (FIG. 21,lanes 1+3).

2.3.2 Knock Down of MZB1 Enhances Proliferation but does not InfluenceApoptosis

Right after establishing the different lines in cell culture, it wasobserved that the shRNA cells proliferated significantly faster comparedto their wt and “rescued” counterparts, making it necessary to splitthem more often. In order to directly assess the role of MZB1 in cellproliferation, growth assays with wt, shRNA and shRNA+MZB1 cells wereperformed. Equal numbers of cells (1.0×10⁵ cells per well) were platedand incubated for 72 hours at 37° C. following the previously describedcell culture protocol. Cell numbers were determined using a CASY® cellcounter (CASY®-technology) after 0 hours, 26 hours and 72 hours afterplating (FIG. 23).

As depicted in FIG. 23, 26 hours after plating, when the culture waspresumably at the end of the lag-phase of growth, there was a slightlyhigher number of shRNA cells than the wt and shRNA+MZB1 cells. After 72hours in culture, in the middle of the exponential growth phase, therewere significantly more shRNA cells than wt (3-4 fold) and shRNA+MZB1cells (˜6 fold). These results indicate that the reduction in MZB1protein level in a fast proliferating B cell line either increases thenumber of cell divisions or decreases the rate of apoptosis. Since thedetermination of cell numbers using the CASY® cell counter(CASY®-technology) did not reveal a change in the percentage of deadcells between the three tested lines (data not shown), the reduction ofMZB1 protein most probably affects proliferation. This hypothesis wouldbe consistent with the fact that the scaffolding protein RACK1, whichinteracts with MZB1 in the membrane fraction of K46 cells (FIG. 9), wasshown to play a central role in the organization of critical biologicalprocesses, such as the regulation of proliferation and cell cycleprogression (McCahill et al., 2002).

In order to verify the results obtained from the cell growth experimentpresented in FIG. 23, cell proliferation was additionally measured using[³H]thymidine incorporation. BCR-stimulated as well as non-stimulatedwt, shRNA and shRNA+MZB1 cells were pulse-labeled with 1.5 μCi/well of[³H]thymidine for 16 h, and the mean [³H]thymidine incorporation oftriplicate cultures was assayed by scintillation counting.

Confirming the results of the growth assay (FIG. 23), non-stimulated K46shRNA cells had an increased rate of proliferation compared to wt (˜8fold) and shRNA+MZB1 (˜3 fold) cells (FIG. 24). Upon BCR stimulation bythe addition of 5 μg/ml α-kappa antibody (Southern Biotechnology), wt,shRNA and shRNA+MZB1 cells responded with equal proliferation rates tothe stimulus. Whereas the wt and the “rescued” cells displayed aremarkable increase in cell proliferation upon BCR stimulation, the cellproliferation rate of K46 shRNA cells stayed almost unchanged (FIG. 24).In other words, non-stimulated K46 shRNA cells exhibit a comparably highproliferation rate like BCR-stimulated wt and “rescued” cells,suggesting a possible pre-activated state of cells with reduced MZB1protein levels. Furthermore, both the growth assay as well as the[³H]thymidine incorporation experiments demonstrate a restoration of thewt proliferation phenotype in shRNA cells with re-established MZB1protein levels (shRNA+MZB1 cells) (FIGS. 23 & 24).

Newly thawed clones of K46 wt cells stably transfected withpSuper.neo+GFP and shRNA cells stably transfected withpSuper.neo+GFP+inv2 were monitored by FACS and the GFP⁺ cells wereenriched by FACS sorting. A representative FACS profile for freshlythawed wt and shRNA cells is depicted in FIG. 25. Generally, only theGFP^(high) cells, from both K46 wt and K46 shRNA cells, are FACS sortedand maintained in selection media. For certain experiments, only theGFP^(high) wt cells were isolated, whereas both the GFP^(high) and theGFP^(intermediate) shRNA cells were sorted and maintained separately(FIG. 25). The GFP^(high) cells (shRNA-GFP-high) should have a morepronounced reduction in MZB1 protein level than the GFP^(intermediate)cells (shRNA-GFP-int cells).

Indeed, an immunoblot analysis carried out with α-MZB1 specificantibodies reveals a correlation between GFP-expression andMZB1-attenuation in the two isolated and separately maintainedpopulations of shRNA cells. Whereas the shRNA-GFP-high cells show amarked reduction in MZB1 protein level, the shRNA-GFP-int populationdisplays a midway down regulation of MZB1 protein (FIG. 26). Thereduction of MZB1 protein level in the shRNA-GFP-high cells in thisparticular experiment is not as pronounced as in the shRNA cells usedfor other analyses (FIG. 21). This might be caused by contamination ofthe shRNA-GFP-high cells with shRNA-GFP-int cells due to the closeproximity of the two gates.

To address the question whether MZB1 influences cell proliferation in adosage-dependent manner, [³H]thymidine incorporation experiments withnon-stimulated as well as BCR-stimulated wt, shRNA-GFP-int andshRNA-GFP-high cells were performed as described above. In thenon-stimulated situation, shRNA-GFP-high cells cycled 2-3 fold fastercompared to the K46 wt cells (FIG. 27). The proliferation rate of thenon-stimulated shRNA-GFP-int cells was in between the that of the wt andthe shRNA-GFP-high cells (FIG. 27). These results indicate adosage-dependent effect of the MZB1 protein on cell proliferation andconfirm the finding that the reduction of MZB1 in non-stimulated B cellspromotes cell division (FIGS. 23, 24 and 27). Similarly to previousobservation (FIG. 24), the proliferation rate of shRNA-GFP-high cellsstayed nearly constant upon BCR stimulation by the addition of α-kappaantibody (Southern Biotechnology) in a concentration of 5 μg/ml. Incontrast, both the wt and the shRNA-GFP-int cells displayed an increasein proliferation upon α-kappa stimulation (FIG. 27). The increase wasmore prominent in case of the wt cells (2-3 fold) than shRNA-GFP-intcells (1.5 fold) (FIG. 27). These results further strengthen the notionthat cells with reduced MZB1 protein levels are in a pre-activatedstate. Lastly, the fact that the reduction of MZB1 protein level in theshRNA-GFP-high cells in the present experiment was not as pronounced asin shRNA cells in the previous experiment (FIG. 24) most likely accountsfor the reduced difference observed in proliferation rate between wt andshRNA cells in the present experiment (FIG. 24: 8 fold vs. FIG. 27: 2-3fold).

In order to confirm that MZB1 influences proliferation and notapoptosis, K46 wt and K46 shRNA cells were stained with Annexin V and7-amino-actinomysin (7-AAD).

Annexin V staining of non-stimulated K46 wt and shRNA cells revealed nosignificant difference in apoptosis (FIG. 28). The majority of cells(approximately 90%) is viable, negative for both Annexin V and 7AAD. Asmall population of cells, ranging from 5% (shRNA) to 6% (wt) areslightly Annexin V-positive, indicating that these cells entered theearly stages of apoptosis. The percentage of dead cells lies in a rangeof 2% in both the wt and the shRNA populations. These results indicatethat a reduction in the MZB1 protein level in K46 mature B cells, atleast in the non-stimulated situation, does not affect apoptosis.

In summary, the RNA interference-mediated downregulation of MZB1expression in K46 mature B cells led to an increase in cell division(FIG. 23, 24 and 27) without affecting apoptosis (FIG. 28). Upondownregulation of MZB1 protein level, the cells exhibited apre-activated state displaying a proliferation rate which is comparableto BCR-stimulated wt K46 cells (FIG. 24). Moreover, thishyperproliferative, pre-activated phenotype appeared to be MZB1dosage-dependent, being more pronounced in cells expressing less MZB1(FIG. 27). Furthermore, MZB1 interacts with RACK1, a scaffolding proteincoordinating the interaction of key signaling molecules such as c-Srcand other Src-family kinases (McCahill et al., 2002), and with PKCβwhich is a critical component of the BCR signaling machinery (Leitges etal., 1996). MZB1 is likely brought into to the BCR signaling machineryvia the formation of a ternary complex with RACK1 and PKCβ, therebyexerting its influence on proliferation and cell cycle progression.Taken together, MZB1 plays a role in the regulation of cellproliferation in non-stimulated K46 mature B cells.

2.3.3 Downregulation of MZB1 Expression Increases Calcium Signalling

In resting B cells, the concentration of free intracellular Ca²⁺ is keptlow and constant. Engagement of the BCR, and hence activation of thecells, results in the recruitment of adaptor molecules such as SLP65 andkinases like Lyn, Syk or Btk, which ultimately results in tyrosinephosphorylation of phospholipase C-γ (PLC-γ) and an increase inintracellular Ca²⁺. The key step in triggering Ca²⁺ flux is theactivation of PLC-γ, which hydrolyzesphosphatidylinositol-4,5-bisphosphate (PIP₂) to diacylglycerol (DAG) andinositol-1,4,5-trisphosphate (IP₃). IP₃ binds to IP₃ receptors (IP₃Rs)in the endoplasmic reticulum (ER) and induces the release of Ca²⁺ intothe cytoplasm. The depletion of Ca²⁺ from intracellular stores triggersentry of Ca²⁺ across channels in the plasma membrane, commonly referredto as calcium release-activated Ca²⁺ (CRAC) channels or store-operatedchannels. The ensuing sustained intracellular Ca²⁺ elevation activatestranscriptional pathways required for proliferation and effector immunefunction, both of which are hallmarks of activated lymphocytes. Theintracellular Ca²⁺ is hence a benchmark for the strength of the receivedsignal and consequently an indicator for the cellular activation status.Since the reduction of MZB1 protein level in K46 mature B cells wasshown to increase proliferation which is a characteristic ofBCR-activated cells, and since MZB1 is likely associated with the BCRsignaling machinery via its interaction partners RACK1 and PKCβ, it ishypothesized that MZB1 also affects calcium signaling. Consequently,Ca²⁺ release following BCR engagement was compared in wt, shRNA andshRNA+MZB1 K46 cells. Relative levels of intracellular Ca²⁺ weremeasured by FACS analysis of Indo-1 AM (Molecular Probes) loaded cells.Ca²⁺-response was induced by adding mouse specific goat α-kappa antibody(Southern Biotechnology) at a final concentration of 5 μg/ml. TheCa²⁺-concentration of the RPMI-media used was either unchanged (2 mM) ordiluted to 0.5 mM in order to simulate limited extracellular Ca²⁺availability.

Under conditions with restricted extracellular Ca²⁺ (0.5 mM), asignificant increase in Ca²⁺ mobilization upon BCR stimulation wasobserved in MZB1 shRNA cells compared to wt and shRNA+MZB1 cells (FIG.29). The wt and the shRNA+MZB1 cells exhibited comparable BCRstimulation-induced Ca²⁺ flux, demonstrating the restoration of the wtphenotype in shRNA cells by re-establishing MZB1 protein level (FIG.29). Similar results were obtained under non-limiting extracellular Ca²⁺conditions (2.0 mM) (FIG. 30).

In order to rule out the possibility that the reduction of MZB1 proteinlevels in K46 cells, which express membrane bound IgG_(2a)/kappa (Kim etal., 1979), increases the amount of cell-surface BCR molecules whichthen leads to the observed increase in Ca²⁺ mobilization, surface BCRexpression in K46 wt and K46 shRNA cells was monitored. The analysis wasperformed using the same freshly thawed clones presented in FIG. 25.

Almost all GFP⁺ K46 wt cells express a kappa-light chain-containing BCRon their cell surface; in contrast, only 70% of the shRNA cells arekappa⁺ (FIG. 31). Whether the remaining 30% of the shRNA cells expressno BCR on their surface and whether the interaction of MZB1 with thelumenal ER chaperone BiP is responsible for this phenotype need to befurther investigated. The fact that the mean fluorescence intensity ofthe kappa⁺ populations among the wt and the shRNA cells is comparablerules out the possibility that the increased Ca⁺ mobilization in theshRNA cells is caused by a higher number of surface BCRs.

In summary, a decrease in MZB1 protein level leads to an increase inCa²⁺ flux upon BCR engagement in K46 mature B cells (FIG. 29, 30),indicating that the reduction of MZB1 expression prompts a morepronounced intracellular signal and hence a stronger activation of thecell following BCR ligation. Together with the results from theproliferation experiments described above, these results suggest thatMZB1 plays a negative regulatory role in BCR-mediated signaling, likelyvia its interaction with RACK1 and PKCβ.

2.3.4 MZB1 Acts as a Potential Regulator of Intracellular Ca²⁺ Stores inK46 Cells

As studies with K46 MZB1siRNA cells revealed an influence of MZB1 oncalcium mobilization following BCR stimulation, a subsequent set ofexperiments should explore whether MZB1 might participate in the morebasic regulation of ER calcium stores. To address this issue, storeoperated calcium entry (SOCE) was induced, stimulating K46 wt, siRNA andsiRNA::MZB1 cells with thapsigargin prior to addition of calcium to afinal concentration of 5.0 mM CaCl₂. Thapsigargin is a specificinhibitor of the SERCA ATPase, which refills ER calcium stores and keepsthe ER calcium concentration at a significantly higher level compared tothe cytosolic calcium concentration. Due to inhibition of the SERCA pumpthe ER calcium concentration decreases within minutes, Stim proteins areactivated and calcium is taken up into the cell over plasma membranelocated calcium release-activated Ca²⁺ (CRAG) channels, which areregulated by Stim proteins. FIG. 32 shows the Ca²⁺-mobilization inMZB1-siRNA cells upon thapsigargin treatment. As seen in FIG. 32, thethapsigargin induced SOCE is significantly higher in K46 siRNA cellscompared to K46 wt and K46 siRNA::MZB1 cells, suggesting that MZB1 mightbe part of the machinery regulating cellular calcium flux (FIG. 32).This notion is further corroborated by the fact that the MZB1 proteinseems to behave similar like Stim proteins, aggregating at ER-plasmamembrane junctions following a thapsigargin-induced decrease in ERcalcium concentration, which could be shown by live cell imaging usingMZB1-GFP fusion protein expressing NIH3T3 fibroblasts: FIGS. 33 and 34show that MZB1 redistributes into punctuate structures after ER Ca²⁺store depletion. Furthermore, the measurement of ER calciumconcentration, using the calcium ionophore ionomycin, revealed asignificant increase in ER Ca²⁺ stores in K46 siRNA cells, compared toK46 wt and K46 siRNA::MZB1 cells, confirming the potential role of MZB1as a modulator of cellular calcium stores, especially ER calcium stores(FIG. 35: Mobilization of ER-Ca²⁺ stores in MZB1-siRNA cells uponionomycin treatment). Interestingly, if the MZB1 protein isoverexpressed in FO B cells, the ER calcium store is found decreased incomparison to MOCK-transduced FO B cells (FIG. 36: Mobilization ofER-Ca²⁺ stores in MZB1-overexpressing FO B cells upon ionomycintreatment), supporting the data observed in the knockdown-studies.

2.3.5 Downregulation of MZB1 Reduces Antibody Secretion in B1/MZ B Cells

Primary B1 B cells and MZ B cells were transduced with retrovirusGP+E86-miRNA-MZB1 or GP+E86-MCS; the transduced cells were enriched byFACS sorting for GFP⁺ cells. MZB1 expression level in the transducedcells was determined using a MZB1-specific antibody in an immunoblotanalysis. MZB1 expression level was found to be reduced in cellstransduced by GP+E86-miRNA-MZB1 (“the miRNA-cells”) than cellstransduced by GP+E86-MCS (“the control cells”) (data not shown). IgMproduction from LPS-stimulated miRNA cells and the control cells isdetermined by ELISA.

2.3.6 Generation and Characterization of MZB1 Knock-Out and ConditionalKnock-Out Mice

MZB1 knock-out (KO) and conditional knock-out mice can be used to studythe role of MZB1 in the regulation of BCR signalling and cellproliferation. The proliferation of non-stimulated MZ and B-1 B cellsfrom wild-type (WT) and knock-out (KO) mice can be determined in a[³H]-thymidine incorporation experiment and compared. Furthermore, Ca²⁺release following BCR stimulation can be measured in MZ and B-1 B cellpopulations obtained from KO and WT animals. MZB1 KO and conditional KOmice can also be used to investigate the differences between MZ and B1 Bcells on the one hand and FO B cells on the other hand. The effect ofthe inactivation of the mzb1-gene on the development and/or homeostasisof MZ and/or B-1 B cells can also be examined. Furthermore, the analysisof MZB1 KO and conditional KO animals will also shine light onto therole of MZB1 in autoimmunity, in particular, the secretion of(auto-)antibodies.

2.4 Functional Analysis of MZB1: Gain of Function 2.4.1 Overexpressionof MZB1 in Follicular B Cells Results in Reduced Proliferation

Follicular (FO) B cells were isolated from 1.5-4 month old miceaccording to their cell surface markers as B220⁺, CD21^(Int) andCD23^(hi) cells by FACS sorting, following standard protocols. Theisolated follicular B cells were resuspended in splenocyte mediacontaining 2 μg/ml polybrene and co-cultured for 36 h on a confluentpackaging cell feeder-layer, either producing an MZB1- andGFP-expressing (GP+E86-MZB1) or a solely GFP-expressing retrovirus(GP+E86-MCS; MOCK control). Transduced, GFP⁺ FO B cells were enriched byFACS-sorting.

An immunoblot analysis carried out with MZB1- as well as GAPDH-specificantibodies revealed a significant MZB1-overexpression in FO B cellstransduced with the MZB1-expressing vector in comparison to MOCKtransduced control FO B cells (FIG. 51). In order to address thequestion whether MZB1 overexpression in primary follicular B cellsaffects cell proliferation, [³H]thymidine incorporation assays wereperformed. Briefly, utilizing a 96 well format to determine the relativerate of radioactive [³H]thymidine assimilation, reflecting the rate ofproliferation, non-stimulated MZB1- and MOCK-transduced follicular Bcells were pulse-labeled with 1.5 μCi/well for 18 h and the mean[³H]incorporation of triplicate cultures was assayed by scintillationcounting.

In non-stimulated primary B cells, MZB1 overexpression resulted in anapproximately three fold downregulation of the proliferation rate (FIG.52), which is consistent with the results of the loss of functionanalyses using shRNA and miRNA. These results suggest that MZB1 is aputative negative regulator of BCR-signaling and as a consequence,influences cell proliferation in the absence of BCR engagement.

2.4.2 Overexpression of MZB1 in Follicular B Cells Results in EnhancedSecretion of IgM Upon LPS Stimulation In Vitro

Follicular B cells that normally express very low levels of MZB1 proteinwere forced to express MZB1 robustly via retroviral infection. Uponinfection, the cells were stimulated with LPS that drives B celldifferentiation into antibody secreting cells. The levels of secretedIgM were assessed in the cell culture supernatant using an ELISA assay.

Significantly higher secretion of IgM was observed on the third day ofstimulation with LPS (FIG. 38). The third day of plasma celldifferentiation in vitro is the time when most of the cells havedifferentiated into antibody secreting cells and the antibody secretionis the highest.

This result suggests that MZB1 positively regulates antibody secretionin B cells.

2.4.3 Generation and Characterization of MZB1 Transgenic Mice

The expression level of MZB1 varies during B cell development indifferent B cell subsets. The highest expression of MZB1 is found inmarginal zone (MZ) B cells and B1 B cells. Very low expression isobserved in resting follicular (FO) B cells. However, the level of MZB1expression increases drastically upon the onset of plasma cellsdifferentiation of FO B cells. Two transgenic mouse models weregenerated, one in which MZB1 overexpression is constitutive and theother in which MZB1 overexpression is inducible, to study the effects ofMZB1 overexpression on B cell development, B cell function, B cellhomeostatsis, in particular, self-renewal, and the development ofautoimmunity.

To generate transgenic mice with constitutive MZB1 overexpression, atransgene construct containing the MZB1 coding sequence under thecontrol of B29 promoter and Eμ enhancer which are active in B cells islinearized and microinjected into the pronuclei of fertilized eggs.Alternatively, a transgene construct containing the MZB1 coding sequenceunder the control of the kappa chain promoter is used.

To generate transgenic mice with inducible MZB1 overexpression, twostrains of transgenic mice were used. One transgenic strain, NLS-TmA,carries a transgene containing the Tet transactivator constitutivelyexpressed from the R26 locus. A second transgenic strain carries atransgene (ptetMZBpa) containing the Tet operator fused upstream to theMZB1 coding sequence. The two transgenic strains are crossed to obtaindouble transgenic mice. The expression of MZB1 can be induced in thedouble transgenic mice by the administration of 1 μg/ml of doxycycline.

Development, function and homeostasis of different B cells subsets, FO Bcells, MZ B cells, and B1 B cells are examined in MZB1 transgenic andnon-transgenic control mice. FACS analysis reveals the relativeabundance of each subset at different stages of develop as well as thesurface phenotype of the different subsets. Functional assays such asproliferation assays and antibody production assays, in the presence andabsence of stimuli, reveal the function and homeostasis of the differentsubsets. In particular, the role of MZB1 in B cell self-renewal isdetermined. The development of spontaneous or experimentally inducedautoimmunity is monitored in MZB1 transgenic and non-transgenic controlmice, as well as MZB1 transgenic mice crossed to mice which arepredisposed to develop autoimmune conditions.

2.4.4 Generation and Characterization of Transgenic Mice with TCell-Specific MZB1 Expression 2.4.4.1 Generation of Transgenic MiceExpressing MZB1 Under the Control of Ick-Promoter

MZB1 was shown to be expressed not only in B cell tissues, but MZB1protein was as well detected in fetal thymus derived DN2 and DN3 cellsand the not yet committed DN1 T cell precursors originating from adultthymus. In all further T cell populations tested so far, includingimmature as well as mature stages of T cell development, MZB1 expressionwas found to be absent. To investigate the impact of ectopic MZB1expression in peripheral T cells with respect to its possible roles inlymphocyte signaling and the regulation of cell proliferation,transgenic mice were generated in which the MZB1 transgene isspecifically expressed in T cells. Briefly, murine MZB1 cDNA wasinserted into the plck-hGH transgenic vector (Garvin et al., 1990),which contains the proximal promoter of the Ick gene for directing Tcell-specific transcription and a portion of the hGH gene for the properprocessing of mRNA by splicing and polyadenylation. The MZB1 transgenicfragment was microinjected into pronuclei of the oocytes of FVB/N mice.Expression of the MZB1 transgene was detected in three independentfounder lines (M3, F38 and F47) by PCR assays and immunoblot analysis(FIG. 39 and data not shown). All experiments to investigate thephenotype of ectopic MZB1 expression in murine peripheral T cells werecarried out with the three transgenic mouse lines, M3, F38 and F47,after backcrossing them with the inbred strain C57BL/6J (JacksonLaboratories). In every investigation performed in this course,transgenic mice (MZB1-transgene) were compared with their correspondingnon-transgenic littermates, referred to as “wt” (wild type). As onlydata is presented with equal results for all of the three transgeniclines, the data of line M3, if not stated differently, isrepresentatively displayed in all further experiments.

In order to examine the expression pattern of ectopically transcribedMZB1 in different T cell subsets isolated from transgenic mice, animmunoblot analysis with MZB1- and GAPDH-specific antibodies wasperformed (FIG. 39) according to standard conditions. 5-6 weeks oldtransgenic mice were sacrificed and thymus derived double negative (DN;CD4⁻ and CD8⁻), double positive (DP; CD4⁺ and CD8⁺) as well as CD4⁺ andCD8⁺ single positive T cells were enriched by FACS sorting according totheir cell surface markers. In addition, CD4⁺ as well as CD8⁺ singlepositive peripheral T cells were isolated from transgenic mice derivedlymph node and spleen, using FACS sorting.

MZB1 protein was shown to be expressed in all of the transgenic T cellsubsets tested, indicating a successful Ick gene-promoter directed, Tcell-specific transcription of the MZB1-transgene (FIG. 39). In afurther analysis, MZB1 expression was examined in thymic, splenic andlymph node-derived single positive T cells originating from transgenicand wt mice. For this set of experiments, sacrificed transgenic mice andtheir corresponding wt littermates were 5-6 weeks of age and theenrichment of CD4⁺ and CD8⁺ single positive T cells was carried outusing FACS sorting.

As depicted in FIG. 40, transgenic single positive T cells expressedMZB1 protein irrespective whether they originated from lymph node,spleen or thymus. In contrast, the analyzed wt derived T cells did notreveal any MZB1 expression. In summary, these results demonstrate thesuccessful generation of transgenic mouse lines which ectopicallyexpress MZB1 in DN and DP thymic precursors as well as thymic, splenicand lymph node single positive T cells.

2.4.4.2 Ectopic MZB1 Expression in Peripheral T Cells Reveals a Changein Intracellular Ceramide Metabolism 2.4.4.2.1 Decreased IntracellularCeramide Content in Transgenic Peripheral T Cells

The siRNA-mediated attenuation of MZB1 expression in K46 cells exhibitedan increase in proliferation rate and overall protein tyrosinephosphorylation of non-stimulated cells, as well as a rise in Ca²⁺mobilization following BCR stimulation. These data identify MZB1 as aputative inhibitory protein which might be involved in the regulation ofBCR-signaling. As the lipid second messenger ceramide was shown to beentailed in biological processes like the regulation of proliferationand furthermore was demonstrated to mediate BCR-induced apoptosis inWEHI 231 immature B cells, the question arose whether the ectopicexpression of MZB1 could influence the intracellular ceramide content intransgenic, peripheral T cells.

Intracellular ceramide levels in non-stimulated as well asTCR-stimulated CD4⁺ splenic T cells derived of wt or transgenic micewere quantified by the diacylglycerol (DAG) kinase assay as previouslydescribed (Tonnetti et al., 1999). In brief, FACS sorted CD4⁺splenocytes were left untreated or induced with α-CD28, α-CD3ε or bothfor 36 hours. Cells were harvested and endogenous ceramide was extractedas described in chapter 5.22. Quantification of thesphingomyelin-derived ceramide was accomplished by incubation of thedissolved lipids with Escherichia coli DAG kinase and [γ-³²P]ATP. Theresulting [³²P]ceramide was quantified using a phosphor-imager system(Fuji) and the AlphaImager™-quantification software (Alpha Innotech)following thin-layer chromatography (TLC).

In non-stimulated CD4⁺ splenic T cells, the ectopic expression of MZB1resulted in an approximately two-fold downregulation of theintracellular ceramide content in comparison to wt cells (FIG. 41).Splenocytes of wt origin, responded to TCR stimulation with modestreduction of endogenous ceramide, prompting a maximal 1.5 folddownregulation of the lipid second messenger after induction withα-CD3ε(0.3 μg/ml) and α-CD28 (1.0 μg/ml). In contrast, transgenicsplenocytes replied with a 1.5 fold reduction of intracellular ceramideto stimulation with α-CD3ε (0.3 μg/ml) followed by a further decrease inceramide content (3.5 fold compared to non-stimulated), if α-CD28 (1.0μg/ml), providing a co-stimulatory signal, was added in addition (FIG.41). In α-CD3ε (0.3 μg/ml) and α-CD28 (1.0 μg/ml) stimulated wt andtransgenic splenocytes, the endogenous ceramide levels in transgeniccells were found to be reduced by a factor of 4-4.5 fold compared to wtT cells. The results presented in FIG. 41 indicate that ectopicexpression of MZB1 in peripheral CD4⁺ T cells causes a decrease inintracellular ceramide levels in the absence of a stimulus. This effectis even more pronounced if cells receive a TCR signal in combinationwith a co-stimulatory, secondary signal mediated by CD28.

In order to investigate whether the observed effects on ceramidemetabolism upon ectopic MZB1 expression might be restricted tolymphocytes, ceramide quantification experiments were performed using aNIH 3T3 murine fibroblast cell line stably expressing FLAG-MZB1 (NIH3T3::FLAG-MZB1). NIH 3T3 as well as NIH 3T3::FLAG-MZB1 fibroblasts weregrown to confluence in the absence of stimulation and harvested.Intracellular ceramide was extracted and quantified as described above.

The non-stimulated fibroblastic cells, irrespective whether MZB1 isexpressed or not, contained equal amounts of intracellular ceramide(FIG. 42), suggesting that the ceramide reduction observed in transgenicT cells might be due to a lymphocyte-specific role of MZB1. This wouldbe in accordance with its expression pattern, being predominantlypresent in early T cell precursors as well as the MZ and B-1 B cellsubsets. Furthermore, data obtained from size exclusion chromatographyindicated that MZB1 is part of a 220 kDa complex in lymphoid cells butnot in NIH 3T3 fibroblasts (data not shown), supporting the notion thatinteraction partners or other factors required for MZB1 function may bemissing in NIH 3T3 cells.

In summary these experiments indicate that ectopic expression of MZB1 innon-stimulated CD4⁺ splenocytes resulted in a reduction of endogenousceramide amounts but had no effect in NIH 3T3 fibroblasts. Aco-stimulatory signal in combination with TCR-engagement furtheraugments these effects, indicating that ectopically expressed MZB1 islikely associated with TCR-signaling in peripheral T cells.

2.4.4.2.2 Unchanged Activity of Acid as Well as NeutralSphingomyelinases in Transgenic Peripheral T Cells

Sphingomyelinases are enzymes that catalyze the hydrolysis ofsphingomyelin (ceramide phosphorylcholin) into ceramide andphosphorylcholin. The reaction is formally similar to that of aphospholipase C. These enzymes appear to be especially interesting asbeing involved in the sphingomyelin signal transduction pathway,mediating signals e.g. by virtue of the lipid second messenger ceramideand regulating cellular processes like apoptosis, cell differentiationand cell proliferation. Sphingomyelinases, which are thought to be,apart from de novo synthesis, a major source of intracellular ceramideare classified according to their pH optimum into five categories,namely the acid sphingomyelinases (A-SMases), the secretorysphingomyelinases (S-SMases), neutral sphingomyelinases (N-SMases; canbe subdivided into Mg²⁺-dependent and -independent), alkalinesphingomyelinases (B-SMases) and the group of bacterialsphingomyelinase-phospholipase C enzymes. In contrast to the otherclasses of SMases, A-SMase and N-SMase participate in signaltransduction and promote, due to their rapid activation by diversestress stimuli, an increase in cellular ceramide levels over a period ofminutes to hours. It was shown that ectopic exression of MZB1 inperipheral T cells decreased intracellular ceramide levels innon-stimulated and, even more pronounced, in TCR-stimulated cells,raising the possibility that MZB1 might regulate the activity of SMases.

The quantification of SMase activity was performed according to aprotocol previously described by Wiegmann and coworkers (Wiegmann etal., 1994). In short, MACS®-purified CD4⁺ splenic T cells of wt andtransgenic origin were cultured in the absence or in the presence ofα-CD3ε (0.3 μg/ml) and α-CD28 (1.0 μg/ml). After 36 h, non-stimulatedand TCR-induced cells were harvested, lysed and the cellular extracts(50 μg total protein) were incubated in the appropriate bufferconditions, being specified by a pH of 5.0 for acid- and a pH of 7.4 forneutral-SMases. Prior to incubation, [N-methyl-¹⁴C]sphingomyelin(Amersham/GE Healthcare) as radioactively labeled substrate was added.The amount of C¹⁴-labeled phosphorylcholine produced from[¹⁴C]sphingomyelin was measured by scintillation counting.

As depicted in FIG. 43, the ectopic expression of MZB1 in CD4⁺peripheral T cells seems to have no impact on A-SMase activity,irrespective whether cells were TCR-induced or left untreated. Moreover,A-SMase activity stayed rather unchanged following TCR stimulation,suggesting that acid-SMase appears not to be involved in TCR-signaling.In a second set of experiments, no change in A-SMase activity between wtand transgenic cells could be observed (FIG. 44).

In contrast to the results presented for acid-SMase activity, themeasured turnover rate of neutral-SMase was found significantlydecreased for both wt and transgenic cells following TCR stimulation.These results indicate that TCR-signaling in combination with aco-stimulatory signal (CD28) decreases N-SMase activity in general,irrespective whether MZB1 is expressed or not.

These findings suggest that transgenic expression of MZB1 in peripheralT cells has no impact on the activity of either acid- or neutral-SMases.Since sphingolipid metabolism is very complex, involving severalalternative routes for the anabolism as well as the catabolism of thelipid second messenger ceramide, the decrease in intracellular ceramidelevels observed in transgenic CD4⁺ T cells might be attributed to theinfluence of ectopically expressed MZB1 on enzymes other than acid- orneutral-SMase. Further experiments examining the activity of e.g.ceramidases, ceramide synthase or other classes of SMases will beperformed in order to explain the reduced ceramide levels detected intransgenic T cells.

2.4.4.3 Transgenic Expression of MZB1 Increases Proliferation inTCR-Stimulated Peripheral T Cells

The intracellular ceramide content, which was shown to beanti-proliferative and pro-apoptotic if increased by agonist- andstress-induced signals, was demonstrated to be decreased especially inTCR-stimulated peripheral T cells ectopically expressing MZB1. Theseresults suggest a possible impact of transgenic transcribed MZB1 onproliferation and/or apoptosis, operating as a pro-proliferative and/oran anti-apoptotic factor, mediating a reduction of endogenous ceramidelevels. To test this hypothesis, in a first series of experiments, theproliferative capacity of transgenic peripheral T cells was compared tothe corresponding wt controls. Cell proliferation was measured using[³H]thymidine incorporation as described previously. In brief, utilizinga 96 well format to determine the relative rate of radioactive[³H]thymidine assimilation, reflecting the rate of proliferation,TCR-stimulated as well as non-stimulated wt, and transgenic CD4⁺ T cellsderived from lymph node or spleen were pulse-labeled with 1.5 μCi[³H]thymidine/well for 18 h and the mean [³H]incorporation of triplicatecultures was assayed by scintillation counting.

As depicted in FIG. 45, α-CD28-stimulated wt and transgenic CD4⁺ lymphnode T cells proliferated equally fast, while transgenic CD4⁺ T cellsdivided 1.5-fold faster following α-CD3ε-stimulation and twice as fastif left untreated. Taking into account that the measured proliferationrate for all of the samples, treated with either exclusively α-CD28 orexclusively α-CD3ε or none, was found to be at low level, the observeddifferences in proliferation rate have to be considered as modest. Incontrast, if stimulated with both α-CD28 and α-CD3ε, transgenic CD4⁺ Tcells proliferated 6.5-fold faster than the wt control, consistent withthe results from the ceramide-assays in which the most pronounceddifference between wt and transgenic cells was observed in CD28- andCD3ε-double-stimulated T cells. Compared to the wt control, transgenic Tcells seemed to be more sensitive to α-CD28 stimulation in the presenceof an α-CD3ε-mediated TCR signal. Whereas the wt T cells revealed noincrease in proliferation when stimulated with α-CD28 in addition toα-CD3ε, the transgenic CD4⁺ T cells did (FIG. 45).

In order to confirm the above results, an analogous series ofexperiments was performed with wt as well as transgenic CD4⁺splenocytes. Similar to lymph node derived T cells, both wt andtransgenic CD4⁺ splenocytes exhibited an unchanged to modest differencein proliferation rate if left untreated, or stimulated with eitherα-CD3ε or α-CD28 (FIG. 46). If CD4⁺ splenocytes were stimulated withboth α-CD3ε and α-CD28, transgenic T cells exhibited a significantincrease in cell proliferation (5.5 fold) in comparison to wt control(FIG. 46). These results confirm the notion that ectopic expression ofMZB1 in peripheral T cells, irrespective if derived from spleen or lymphnode, results in a cellular state of changed responsiveness toTCR-ligation in conjunction with a co-stimulatory signal provided byCD28 (FIGS. 45 and 46).

The [³H]thymidine incorporation experiments were repeated as well withan increased α-CD28-(0.3 μg/ml) but an unchanged α-CD3ε-concentration(0.3 μg/ml), exhibiting comparable results for splenic as well as lymphnode derived CD4⁺ T cells (FIG. 47).

The stimulation with both α-CD3ε (0.3 μg/ml) and α-CD28 (0.3 μg/ml),resulted, in the case of CD4⁺ transgenic T cells, in an approximately 4fold increase in proliferation compared to α-CD3ε-single stimulatedcells and a 20 fold higher [³H]thymidine incorporation compared to theuntreated control (FIG. 47). The results for both lymph node and splenicCD4⁺ T cells are similar to those obtained in the previous experimentsusing α-CD28 at a concentration of 0.1 μg/ml (FIGS. 45 and 46). Thesedata indicate that the conditions chosen in the first set ofexperiments, 0.3 μg/ml of α-CD3ε and 0.1 μg/ml of α-CD28, offeredmaximal stimulation to transgenic T cells. In contrast, upon stimulationof the TCR in conjunction with a co-stimulatory signal, using the lowerα-CD28 concentration (0.1 μg/ml), wt CD4⁺ T cells did not exhibit afurther increase in proliferation compared to α-CD3ε-single stimulatedcells (FIGS. 45 and 46). Only when α-CD28 concentration was raised to0.3 μg/ml, did wt T cells respond to stimulation with both α-CD3ε (0.3μg/ml) and α-CD28 (0.3 μg/ml) with an approximately 3 fold increase inproliferation compared to α-CD3ε-single stimulated wt cells (FIG. 47).Moreover, if induced with α-CD3ε (0.3 μg/ml) and the higherconcentration of α-CD28 (0.3 μg/ml), splenic as well as lymph nodederived transgenic T cells displayed a 2.5 fold faster proliferationrate compared to the wt controls (FIG. 47), which is consistent with theresults obtained in the first series of experiments, revealing a 5.5 to6.5 fold increased in [³H]thymidine incorporation in TCR and α-CD28 (0.1μg/ml) stimulated transgenic T cells (FIGS. 45 and 46). This reductionfrom a 6.5 fold (low α-CD28 concentration) to a 2.5 fold (high α-CD28concentration) difference in proliferation, observed in the secondseries of experiments might be attributed to the changed conditions,using an increased α-CD28 (0.3 μg/ml) concentration.

In summary, these results suggest that transgenic peripheral T cellsrespond to TCR-signaling, especially when provided with a co-stimulatorysignal, with a significantly increased proliferation rate in comparisonto wt T cells. Moreover, compared to wt controls, transgenic T cellsresponsed to a lower concentration of α-CD28 (0.1 μg/ml), suggestingthat ectopic MZB1 expression enhanced the reactiveness of T cellstowards TCR-signaling.

In order to show that the effects observed in [³H]thymidineincorporation experiments are due to an impact of ectopically expressedMZB1 on proliferation and not apoptosis, TdT-mediated dUTP nick endlabeling (TUNEL) assays with stimulated wt and transgenic, peripheral Tcells were performed.

As depicted in FIG. 48, the TUNEL assay performed with TCR-stimulated wtand transgenic peripheral T cells of splenic origin revealed no obviousdifferences between the tested samples with respect to apoptosis. After24 hours of stimulation with either α-CD3ε (0.3 μg/ml) or α-CD3ε (0.3μg/ml) in conjunction with α-CD28 (0.1 μg/ml), the vast majority ofcells (>95%), whether isolated from wt or transgenic animals, remainedTUNEL negative (FIG. 48). In the wt control, between 3.5% (α-CD3ε- andα-CD28-) and 4.9% (α-CD3ε) of the cells entered the final stages ofapoptosis. In case of the transgenic T cells, the numbers were slightlyreduced and ranged between 2.7% (α-CD3ε- and α-CD28) and 2.8% (α-CD3ε).This suggest that ectopic expression of MZB1 in peripheral T cellsexerts only very modest influences on apoptosis.

In summary, ectopically expressed MZB1 has an effect on cellproliferation in TCR-stimulated CD4⁺ peripheral T cells; apoptosis doesnot seem to be affected. However, since the percentage of apoptoticcells detected following 24 hours of TCR stimulation was very low inboth the wt and the transgenic cells (FIG. 48), further conditionsinducing more pronounced programmed cell death will be tested.

2.4.4.4 Transgenic MZB1 Expression in Peripheral T Cells AugmentsIntracellular Calcium Release Upon TCR Stimulation

The basic steps of BCR and TCR signal transduction proceed in a fairlyparallel way. Similar to resting B cells, the concentration of freeintracellular Ca²⁺ is kept low and constant in non-stimulated T cells.Engagement of the TCR results in the recruitment of adaptor moleculesand kinases, ultimately leading to tyrosine phosphorylation of PLC-γ andan increase in intracellular Ca²⁺. The activation of PLC-γ prompts thegeneration of DAG and IP₃, leading to the depletion of Ca²⁺ fromintracellular stores and finally triggering the entry of Ca²⁺ acrosschannels in the plasma membrane, commonly referred to as calciumrelease-activated Ca²⁺ (CRAC) channels. The ensuing sustainedintracellular Ca²⁺ elevation activates transcriptional pathways requiredfor proliferation and effector immune function, both of which are ahallmark of activated B as well as T cells. Hence, the robustness ofincrease in intracellular Ca²⁺ reflects the strength of the receivedTCR-mediated signal. The ectopic expression of MZB1 in peripheral Tcells led to a decrease in intracellular ceramide levels and an increaseof cell proliferation in T cells stimulated simultaneously with α-CD3εand α-CD28. Since the phenotypes observed for transgenic T cells are themost pronounced when the cells were stimulated by a TCR signal,indicating a possible role of MZB1 as an activator of TCR-signaling. Inorder to address the issue whether ectopic expression of MZB1 inperipheral T cells changes the TCR-signaling capacity of these cells,Ca²⁺ release following TCR-engagement was compared in wt and transgenicCD4⁺ splenocytes. Relative levels of intracellular Ca²⁺ were measured byfluorescence-activated cell sorting (FACS) analysis of Indo-1 AM(Molecular Probes) loaded cells. Ca²⁺-response was either induced byaddition of α-CD3ε at a final concentration of 10 μg/ml or by addingboth, α-CD3ε (10 μg/ml) and α-CD28 (5 μg/ml). The Ca²⁺-concentration ofthe used RPMI-media remained unchanged at a value of 2 mM Ca²⁺.

As depicted in FIG. 49, there were only modest differences in Ca²⁺ fluxof α-CD3ε-stimulated wt and transgenic T cells. In contrast, asignificant increase in Ca²⁺ mobilization in the transgenic cellscompared to wt control was observed when the cells were stimulatedthrough both TCR and CD28 (FIG. 50). These data are consistent with theobserved phenotypes of a marked reduction in intracellular ceramidelevels and a prominently increase in cell proliferation in TCR andCD28-stimulated transgenic T cells compared to wt control cells,suggesting an impact of ectopically expressed MZB1 on the TCR-signalingmachinery. Notably, for all phenotypes associated with transgenic MZB1expression in T cells observed so far, in addition to TCR ligation, aco-stimulatory signal mediated by CD28 is required. Consequently, thepossibility that transgenic MZB1 expression increases the amount ofcell-surface TCR molecules and hence leads to the observed increase inCa²⁺ mobilization can be fairly safely ruled out. Further FACSexperiments will be performed in order to investigate the influence ofectopic MZB1 expression on CD28 surface expression on transgenic Tcells.

Whether the significantly reduced ceramide levels in α-CD3ε- andα-CD28-induced transgenic T cells is be a cause or consequence ofincreased cell proliferation and augmented Ca²⁺ flux, both detected inTCR- and CD28-stimulated cells, will be investigated.

Interestingly, whereas the RNA interference-mediated downregulation ofMZB1 expression in K46 mature B cells suggested a possible role of MZB1as an inhibitory modulator of BCR-signaling, attenuating Btk activityand hence reducing proliferation, tyrosine phosphorylation andCa²⁺-mobilization upon BCR-ligation, ectopically expressed MZB1 seems toplay a different role in T lymphocytes. In the transgenic mice with Tcell-restricted expression of MZB1, the protein appears to act as anactivator of TCR-signaling, resulting in an increased cell proliferationand enhanced Ca²⁺-mobilization following TCR/CD28 stimulation. Thedifference in cell type-specific functions of MZB1 in B and T cellsmight be explained, at least partially, but differences in celltype-specific interaction partners and/or other cellular factors. Tcell-specific interaction partners can be identified by the same methodswhich led to the identification of interaction partners in B cells.

Since MZB1 apparently enhances TCR-mediated signaling (especically inthe presence of co-stimulation through CD28) upon ectopic expression inT cells, activators and enhancers of MZB1 expression may be used toenable expression of MZB1 in T cells, thereby enhancing TCR-mediatedsignalling, in particular, in the presence of co-stimulation, such asthat through CD28. Activators and enhancers of MZB1 expression may beused to enhance TCR-mediated signalling in T cells in vitro or in vivo.In one embodiment, the activators and enhancers of MZB1 expression maybe used to treat diseases which are caused by and/or associated with Tcell malfunctioning due to reduced or absent TCR signalling and/orco-stimulation, such as hereditary, spontaneous, and acquiredimmunodeficiencies. Activators and enhancers of MZB1 expression may alsobe used to enhance TCR-mediated signalling in T cells in order toincrease the ease in establishing T cell clones with desired TCRspecificities. For example, transgenic mice expressing MZB1 in T cellscan be immunized with an antigen and antigen-specific T cells clones canbe derived following in vitro stimulation(s). Alternatively, mice nottransgenic for MZB1 can be immunized with an antigen, T cells can beisolated, transfected or transduced with an MZB1 expression vector invitro, and stimulated in vitro through the TCR in order to generate Tcell clones with desired antigen-specificity.

Since MZB1 appears to have different effects on BCR and TCR signallingin B cells and T cells, respectively, for certain applications, inparticular certain in vivo applications, it may be desirable to deliveractivators and/or enhancers of MZB1 expression in a cell type-specificmanner.

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1.-30. (canceled)
 31. A method of treating an autoimmune disease in apatient comprising administering an effective amount of an inhibitor ofthe expression or at least one of the biological activities of apolypeptide to a patient with an autoimmune disease, thereby treatingthe autoimmune disease in the patient, wherein the polypeptide comprisesan amino acid sequence selected from the group consisting of: (a) theamino acid sequence as set forth in SEQ ID NO: 4, (b) an amino acidsequence encoded by the nucleotide sequence set forth in SEQ ID NO: 3,(c) an amino acid sequence of (a) or (b) further comprising anamino-terminal methionine, (d) an amino acid sequence which is a humanortholog of any of (a)-(c), optionally further comprising anamino-terminal methionine, (e) an amino acid sequence which is at least72% identical to any of (a)-(d) over its entire length, optionallyfurther comprising an amino-terminal methionine, and wherein (d)-(e) hasat least one of the biological activities of the polypeptide having theamino acid sequence of any of (a)-(c), and wherein the inhibitor is anantisense RNA, siRNA, shRNA, a ribozyme, a RNA or DNA aptamer, decoyRNA, a peptide aptamer, a small molecule, or an antibody.
 32. The methodof claim 31, wherein the inhibitor is an antibody.
 33. The method ofclaim 32, wherein the antibody is specific for the polypeptide of any of(a)-(e).
 34. The method of claim 33, wherein the antibody is specificfor the polypeptide of any of (a)-(d). 35.-39. (canceled)
 40. The methodof claim 31, wherein the polypeptide is (a).
 41. The method of claim 40,wherein the inhibitor is an antibody.
 42. The method of claim 31,wherein the polypeptide is (b).
 43. The method of claim 42, wherein theinhibitor is an antibody.
 44. The method of claim 31, wherein thepolypeptide is (c).
 45. The method of claim 44, wherein the inhibitor isan antibody.
 46. The method of claim 31, wherein the polypeptide is anamino acid sequence which is a human ortholog of (a), optionally furthercomprising an amino-terminal methionine.
 47. The method of claim 46,wherein the inhibitor is an antibody.
 48. The method of claim 31,wherein the polypeptide is an amino acid sequence which is a humanortholog of (b), optionally further comprising an amino-terminalmethionine.
 49. The method of claim 48, wherein the inhibitor is anantibody.
 50. The method of claim 31, wherein the polypeptide is anamino acid sequence which is a human ortholog of (c), optionally furthercomprising an amino-terminal methionine.
 51. The method of claim 50,wherein the inhibitor is an antibody.
 52. The method of claim 31,wherein the polypeptide is (e).
 53. The method of claim 52, wherein theinhibitor is an antibody.